Rat Marrow Research Articles

Tissue transglutaminase_variant 2-transduced mesenchymal stem cells and their chondrogenic potential.

As cartilage is incapable of self-healing upon severe degeneration because of the lack of blood vessels, cartilage tissue engineering is gaining importance in the treatment of cartilage defects. This study was designed to improve cartilage tissue regeneration by expressing tissue transglutaminase variant 2 (TGM2_v2) in mesenchymal stem cells (MSC) derived from bone marrow of rats. For this purpose, rat MSCs transduced with TGM2_v2 were grown and differentiated on three-dimensional polybutylene succinate (PBSu) and poly-l-lactide (PLLA) blend scaffolds. The transduced cells could not only successfully express the short form transglutaminase-2, but also deposited the protein onto the scaffolds. In addition, they could spontaneously produce cartilage-specific proteins without any chondrogenic induction, suggesting that TGM2_v2 expression provided the cells the ability of chondrogenic differentiation. PBSu:PLLA scaffolds loaded with TGM2_v2 expressing MSCs could be used in repair of articular cartilage defects.

Combined Effects of Irradiation and Hindlimb Suspension on Erythroid Lineage Precursors from Rat Bone Marrow.

Erythroid precursors from the femoral bone marrow of Wistar rats were characterized after 30-day hindlimb suspension, fractionated γ-radiation, and their combination. After hindlimb suspension, the total content of myeloid CFU decreased; activity of erythroid differon also considerably suppressed, which manifested in a decrease in the number of erythroid burst-forming units and area of colonies formed by erythrocyte precursors. After irradiation and combined exposure to these two factors, no significant differences from the control were revealed; optical density of formed colonies slightly increased in all experimental groups. Thus, suppression of the erythroid lineage was most pronounced during hindlimb unloading. The combined effect of radiation and hindlimb suspension produced no appreciable negative effect on erythropoiesis in rat bone marrow.

Chronic Hypoxia-Induced Microvessel Proliferation and Basal Membrane Degradation in the Bone Marrow of Rats Regulated through the IL-6/JAK2/STAT3/MMP-9 Pathway.

Chronic hypoxia (CH) is characterized by long-term hypoxia that is associated with microvessel proliferation and basal membrane (BM) degradation in tissues. The IL-6/JAK2/STAT3/MMP-9 pathway has been described in a variety of human cancers and plays an essential role in microvessel proliferation and BM degradation. Therefore, this study investigated the role of the IL-6/JAK2/STAT3/MMP-9 pathway in hypoxia-mediated microvessel proliferation and BM degradation in the rat bone marrow. Eighty pathogen-free Sprague Dawley male rats were randomly divided into four groups (20 per group)—control group, CH group (exposed to hypoxia in a hypobaric chamber at a simulated altitude of 5000 m for 28 d), CH + STAT3 inhibitor group (7.5 mg/kg/d), and CH + DMSO group. Microvessel density (MVD) and BM degradation in the bone marrow were determined by immunofluorescence staining and transmission electron microscopy. Serum IL-6 levels were assessed by enzyme-linked immunosorbent assay (ELISA), and the levels of P-JAK2, P-STAT3, and MMP-9 were assessed by western blot analysis and real-time reverse transcription PCR (RT-PCR). Hypoxia increased serum IL-6 levels, which in turn increased JAK2 and STAT3 phosphorylation, which subsequently upregulated MMP-9. Overexpression of MMP-9 significantly promoted the elevation of MVD and BM degradation. Inhibition of STAT3 using an inhibitor, SH-4-54, significantly downregulated MMP-9 expression and decreased MVD and BM degradation. Surprisingly, STAT3 inhibition also decreased serum IL-6 levels and JAK2 phosphorylation. Our results suggest that the IL-6/JAK2/STAT3/MMP-9 pathway might be related to CH-induced microvessel proliferation and BM degradation in the bone marrow.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3–10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2–15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

Immunoglobulin G amplifies the production of regulatory rheumatoid factor in vitro through idiotype-anti-idiotype interactions

ABSTRACTImmunoglobulin G can inhibit antibody response. The mechanism of immunosuppression by immunoglobulins remains unknown. Recently, we found a new factor of immunoregulation referred to as regulatory rheumatoid factor (regRF). RegRF prevents autoimmunity and reduces experimental autoimmune reactions. RegRF comprises a population of anti-idiotypic antibodies that have a unique paratope specific to the antigen-binding sites of the antibodies, and a shared paratope specific to neoepitopes of IgG Fc fragments. Given the specificity of regRF, we can anticipate that IgG would be able to induce regRF production, and consequently that the immunosuppressive effect of IgG may be mediated by regRF. We found that IgG induces regRF production in a culture of B lymphocytes obtained from the red bone marrow of intact rats. IgG does not expose neoepitopes recognized by the shared paratope of regRF, and does not acquire them in culture. Therefore, the stimulation of regRF production induced by IgG is not a result of the interaction between the shared paratope of regRF and the neoepitopes of IgG. Fc fragments of IgG are unable to stimulate regRF production. Fab fragments inhibit spontaneous regRF production. F(ab´)2 fragments stimulate regRF production in lymphocyte culture. We theorize that IgG activates regRF-producing lymphocytes through idiotype-anti-idiotype interactions.

Activating α7nAChRs enhances endothelial progenitor cell function partially through the JAK2/STAT3 signaling pathway

Microvascular injury is a common pathological process in ischemia-reperfusion injury. Endothelial progenitor cells (EPCs) are vital cells for angiogenesis and endothelial repair. These cells can home to injury sites and secrete angiogenic growth factors. α7nAChRs are pivotal in cholinergic angiogenesis, which is associated with endothelial cells and EPCs. Our study was designed to determine whether activating α7nAChRs enhances the function of EPCs and to explore the underlying mechanism. EPCs were derived from the bone marrow of male Sprague-Dawley rats and treated with an α7nAChR agonist (PNU282987), an α7nAChR antagonist (MLA) and a JAK2 antagonist (AG490). We then assayed the angiogenic abilities of the EPCs, including proliferation ability, adhesion ability, migration ability and in vitro tube formation ability. The levels of total JAK2 (t-JAK2), phosphorylated JAK2 (p-JAK2), total STAT3 (t-STAT3) and phosphorylated STAT3 (p-STAT3) were estimated by western blot analysis. PNU282987 treatment facilitated the angiogenic abilities of EPCs compared with the control regimen. The western blot data suggested that PNU282987 increased the levels of p-JAK2 and p-STAT3. However, the differences in t-JAK2 levels and t-STAT3 levels between the agonist-treated group and the control group were not significant. Moreover, treating EPCs with AG490 reduced STAT3 phosphorylation and attenuated the PNU282987-induced enhancement of EPCs. We demonstrated that activating α7nAChRs can enhance EPC functions partially through the JAK2/STAT3 signaling pathway. This study reveals that α7nAChRs are potential therapeutic targets for angiogenesis and that the JAK2/STAT3 pathway plays a vital role in the associated therapeutic mechanism.

Histomorphological characterization of Pneumococcal meningitis in bone marrow of Wistar rats treated with Bridelia ferruginea and ciprofloxacin

Background: Bridelia ferruginea is one of the most valuable ethnomedicinal plants, which has been confirmed for antimicrobial activities and tissue-enhancing properties against degenerative processes. This study aimed at investigating the histomorphological characterization of ethanol extracts of leaf, stem-bark, and root of B. ferruginea on Pneumococcal-meningitis-induced bone marrow damage. Materials and Methods: Sixty-three Wistar rats were randomly divided into nine groups of seven rats each. Group B-F2 were inoculated with 1 × 109 colony-forming units (CFU)/mL of P. meningitis and were left for 72h before treatment. Group A rats (normal control) received 2.5 mg/kg of phosphate-buffered saline (PBS), whereas group B rats (negative control) were left untreated, group C rats (positive control) were treated with single dose daily of 1.4 mg/kg ciprofloxacin tablets, group D1 received 300 mg/kg B. ferruginea leaf extract, group D2 received 600 mg/kg B. ferruginea leaf extract, group E1 received 300 mg/kg B. ferruginea stem-bark extract, group E2 received 600 mg/kg B. ferruginea stem-bark extract, group F1 received 300 mg/kg B. ferruginea root extract, and group F2 received 600 mg/kg B. ferruginea root extract for 2 weeks after which all the animals were killed via cervical dislocation. Results: Ciprofloxacin and the leaf, stem-bark, and root extracts of B. ferruginea were able to avert and managed damage (tissue damage, histomorphological, and cytoarchitectural parameter) caused by P. meningitis in the bone marrow of adult Wistar rats. Conclusion: Bridelia ferruginea has antibacterial activities against factors causing bone marrow erythyroid hyperplasia, osteomyelitis as well as hemolytic anemia.

Toxic effect of copper-zinc pyrite ore on erythropoiesis in chronic experiment

Natural ores contain a large number of harmful components for human health. Workers of mining and processing enterprises, who have long-term contact with these natural elements, are often diagnosed with anemia, that pathogenesis is not sufficiently studied. Aim of the study was to detail the mechanisms of rats’ erythron disorders in the long-termintoxication by natural complex of heavy metal compounds of copper-zinc pyrite ore. Material and methods. The work was performed on 60 white non-linear male rats aged 3-4 months weighing 220,52 ± 15,51 g. The experimental group of animals (n = 40) were injected orally with water suspension of copper-zinc powder in a bread crumb an hour before standard feeding during 75-120 days. The blood and bone marrow of experimental groups of rats were carried out on the 75-th, 90-th, 105-th and 120-th days of the experiment. The blood and bone marrow of rats’ control groups were studied on the 75-th and 105-th day. The central part of erythron was assessed by the number and composition of the erythroblastic islets (EI) of bone marrow, the number of free macrophages, the coefficients of involvement of colonyforming units of red blood cells (CFU-E) and macrophages into erythropoiesis. Results . In the peripheral blood of experimental rats the number of red blood cells and the content of hemoglobin were reliably less than the control values on the 90-th and 120-th day, the number of reticulocytes was exceeded the control group on the 75-th, 105-th and 120-th day. In the bone marrow of experimental rats there were only a single EI1 and EI2 classes of maturity. Throughout the experiment, the content of free macrophages and EI with reticulocytes «crown» in the bone marrow of rats was elevated, and the concentration of iron and erythropoietin in the blood, on the contrary, was reduced. The intensity of CFU-E involvement in the erythropoiesis was less than the control level by 2 times on 105th and 120th days of the experiment. Conclusion . With long-term introduction of copper-zinc pyrite ore in the bone marrow of rats the process of formation of EI de novo is suppressed by destabilizing the contact of free bone macrophages with young red blood cells. There is reduces in the content of serum erythropoietin and bound iron, that combines with the change of erythropoiesis nature, leads to decreasing of the number of mature red blood cells and content of hemoglobin in the peripheral blood.

Chitosan channels with stuffed mesenchyme originated stem/progenitor cells for renovate axonal regeneration in complete spinal cord transection

AIM To examine the implantation of chitosan channels stuffed with mesenchyme-originated stem/progenitor cells (MSPCs) derived from adult rats in a spinal cord transection model. The level of axonal regeneration, the effect of chitosan channels on the survival of MSPCs, and the functional recovery results were also evaluated. MATERIAL AND METHODS Chitosan channels stuffed with MSPCs were implanted at the level of T8 in a transected rat spinal cord. MSPCs were harvested from the pelvic bone marrow of adult rats, and the MSPC?chitosan channel group was compared with three control groups. The axonal regeneration capacity, the effect of chitosan channels on the survival of MSPCs, and the functional recovery results were compared among four groups. The survival of MSPCs was evaluated using histopathological techniques and electron microscopy, axonal regeneration/germination was evaluated by confocal microscopy, and locomotor function was assessed for 4 weeks using the Basso, Beattie, and Bresnahan locomotor score. RESULTS The MSPC-chitosan channel group exhibited enhanced survival of transplanted MSPCs compared with MSPCs transplanted directly into the lesion cavity, although no significant difference was detected in locomotor function between the treatment and control groups. The MSPC-chitosan channel group demonstrated thicker myelination of axons than the other groups. CONCLUSION Chitosan channels promoted the survival of transplanted MSPCs and created a tissue bridge after complete spinal cord transection. They also induced axonal regeneration and germination. No significant improvement in functional recovery was found between the groups.

Therapeutic potential of a biomedical cellular product in rats with lower limb ischaemia

Critical ischaemia of lower limbs is a cause of death and invalidity in the whole world. Stem cells and products of their secretion find wide application in treatment of vascular diseases, including critical ischaemia of the lower limbs. Erythropoietin promotes an increase in the angiogenic potential of stem cells. The authors examined the therapeutic potential of a biomedical cellular product (mesenchymal stem cells and products of their secretion) and mesenchymal stem cells with erythropoietin on the processes of restoration of vessels in the hind legs of Wistar male rats following induction of lower limb critical ischaemia. Mesenchymal stem cells were derived from the bone marrow of male Wistar rats. Critical ischaemia of hind legs was modulated by transaction of the femoral artery. The parameters of microcirculation in the foot were assessed with the help of laser Doppler flowmetry. In the blood serum and crural muscles by means of solid-phase enzyme immunoassay we examined the levels of cytokines, growth factors, and persistent metabolites of nitrogen oxide - nitrites. Muscles morphology and the number of blood vessels were assessed by the findings of histological examination. It was shown that the biomedical cellular product alone and in combination with erythropoietin stimulated angiogenesis. The results of Doppler flowmetry revealed restoration of the parameters of microcirculation in the lower limb by 35-75% of the baseline values. Besides, we observed a decrease of muscle necrosis, connective tissue proliferation, and an increase in the number of the vessels supplying the muscles in the experimental groups. It was also determined that the biomedical cellular product influenced the levels of cytokines in blood serum and crural muscles. Hence, the obtained findings proved the therapeutic potential of the biomedical cellular product in critical ischaemia of lower limbs.

Степень активации гемопоэза с помощью суммарной РНК зависит от способа ее парентерального введения

The effects of hematopoiesis-stimulating total RNA were studied with subcutaneous (s/c), intramuscular (i/m), intraperitoneal (i/p), intravenous (i/v) and intranasal (i/n) routes of administration. Total RNA was isolated from the lymphoid cells of the spleens of healthy rats 17 hours after blood loss. Benzene anemia was used as a model of inhibited erythropoiesis. 28 days after the first benzene injection, RNA was introduced into rats. After 7 days in i/p, i/v, i/n groups the number of reticulocytes and platelets increased 1.5-2 times, in i/v and i/n groups the number of leukocytes increased 1.4-1.6 times. After 14 days in i/p, i/v, i/n groups the number of reticulocytes increased 2-2.6 times, the number of leukocytes - 1.6 times, platelets - 1.4-1.6 times. After 21 days in these groups, the number of reticulocytes was 3-4 times higher than the control rats, leukocytes - 1.4 times, platelets - 1.5-1.7 times. After 21 days the effect of the intramuscular administration of RNA appeared: the number of reticulocytes increased 2 times, the number of platelets - 1.2 times. On 21 days in rats were examined myelograms. With all groups, except «s/c», the number of basophilic, polychromatophilic and orthochromatic erythroblasts, as well as the number of megakaryocytes, significantly increased in the bone marrow. The number of myeloblasts, promyelocytes, myelocytes and lymphocytes increased in the bone marrow of rats only in i/p, i/v, i/n groups. Thus, the best methods for introducing total RNA were intraperitoneal, intravenous and intranasal routes of administration. With the intramuscular introduction, the stimulating hematopoiesis effect appeared only by 21 days. Sbcutaneous rout of administration administration of total RNA was ineffective.

Эффективность экстрактов <i>Saussurea controversa</i> и <i>Fillipendula ulmaria</i> на фоне антибиотикотерапии остеомиелита в эксперименте

Курсовое применение экстрактов Saussurea controversa DC (S.c.) и Fillipendula ulmaria (L). Maxim. (F.u.), содержащих широкий набор флавонолгликозидов, при экспериментальном остеомиелите приводит к стимуляции гранулопоэза в 2,8 и 2,2 раза соответственно (p = 0,04) и регенерации костной ткани (плотность распределения остеобластов возрастает в 5 раз, p = 0,02) в зоне поражения у крыс. В результате исследования миелограмм костного мозга отмечены активация митотической активности гранулоцитов, увеличение количества миелобластов в 5,6 и 4,2 раза (p = 0,02) и зрелых форм гранулоцитов (в 2,5 и 1,5 раза, p = 0,04), измененных в результате антибиотикотерапии. После применения экстракта S.c. в костном мозге крыс с остеомиелитом возрастает общее количество ядросодержащих клеток в 2,7 (p = 0,04) раза. Улучшение морфологической картины костной ткани (активация клеток эндоста, периоста, появление остеобластических «почек», формирование грануляционной ткани) и формирование зрелой костной ткани более выражено (на 12 %, p = 0,02) при применении экстракта S.c., по сравнению с экстрактом F.u.

Multiple-endpoint genotoxicity assay for colon carcinogen 1,2-dimethylhydrazine

Human risk assessment of the toxic potency of chemicals typically includes genotoxicity assays for predicting carcinogenicity. Gene mutation frequency and chromosomal aberration are two major genotoxicity endpoints in standardized in vitro and in vivo assays. The weight-of-evidence approach in risk assessment is more focused on in vivo assay results; however, animal welfare considerations are aimed at the reduction, replacement, and refinement (3R’s) of animal experiments, including a reduction in the number of experimental animals. Proposals to reduce experimental animals in genotoxicity testing include the incorporation of genotoxicity endpoint(s) into other toxicological studies and the combination of two or more assays detecting different genotoxicity endpoints in the same animals. In this study, we used 1,2-dimethylhydrazine as a model chemical of colon carcinogen to assess gene mutation frequency and chromosomal aberration in vivo simultaneously. Specifically, a gene mutation frequency assay was combined with a multiple-organ micronucleus test (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic rats. Both gpt mutant frequency and micronucleated cell frequency significantly increased in colon and liver but not in bone marrow. Interestingly, we found that the colon carcinogen induced both gene mutations and micronuclei in the targeted colon tissue. Thus, we demonstrated that the mechanism of a carcinogen could be derived from an animal experiment using a lower number of experimental animals as currently recommended. Moreover, a significant increase in mutant frequency in colon and liver was already observed on the first day after treatment completion, as well as on the third day, which is the guideline-recommended period. Thus, this endpoint is compatible with other genotoxicity assays. We confirmed that performing the micronucleus assay in combination with a gene mutation assay in F344 gpt delta transgenic rats is useful to evaluate different genotoxic endpoints simultaneously in the same animals, which reduces the number of experimental animals.

MSCs ARE LOCATED IN THE PERINEURIUM OF THE RECIPIENT RAT AFTER ALLOTRANSPLANTATION INTO DAMAGED NERVE

The gene and cell therapy, stimulating the regeneration of damaged nerves is currently under development. In these experiments mesenchymal stem cells (MSCs) are often used. The purpose of this study is to describe the localization and morphological features of mesenchymal stem cells derived from bone marrow after their allografting into the damaged rat nerve. MSCs of the bone marrow of Wistar-Kyoto rats were obtained from Transtechnology LLC (Head G.Polyntsev, Ph.D.). MSCs were cultured, identified and labeled by 5-bromo-2’-deoxyuridine (BrdU) in vitro. The sciatic nerve of adult Wistar-Kyoto rats (n = 12) was damaged (ligature, 40 sec), and the suspension of BrdU+ MSCs (5 · 104 cells in 5 μl per animal) was immediately transplanted into the damaged sciatic nerve. In a previous study, we have showed that some transplanted cells are located in the epineurium of the recipient’s nerve. The perineurium of the recipient rats was studied in the present work. Perineurial cells have polygonal form, thin and flat cell nucleus and form several layers, the basement membranes being placed between them. Perineurium is characterized by the presence of occluding junctions that can be identified using anti-claudine antibodies. The use of antibodies to claudine allowed us to identify perineurium. Some BrdU+ MSCs were found to survive 5-7 d following surgery and according to their localization and morphology became perineurium cells. Such extracellular matrix proteins as laminin, fibronectin, collagen are present in the perineurium. Apparently, the presence of these proteins creates a favorable biological environment for MSCs survival and for their differentiation towards perineurium cells. The results of the study confirm the mesenchymal origin of perineurium cells.

Hepatic protein synthesis and morphological parameters in blood of rats under oxidative stress and action of feed additive Butaselmevit-plus

The intensive development of animal husbandry at the present stage requires new approaches to organizing the feeding of farm animals and the introduction of modern feed additives, which are not normally used purely as feed, but are deliberately added to feed or water to improve their quality, increase productivity and animal welfare. To prevent the development of oxidative stress in animals, we used the feed additive "Butaselmevit-plus", which in its composition contains the fruits of milk thistle, selenium, methionine and vitamins A, E and D3. The aim of the study was to investigate the effect of feed additive Butaselmevit-plus on blood morphological parameters and protein synthesis of rat liver under tetrachloromethane poisoning. For research, we used Wistar male rats, which were divided into three groups of 20 animals each. Control rats were intact. The first and second experimental group of rats were administered tetrachloromethane. Experimental intoxication in animals was performed by twice (after 48 h) intragastric administration of tetrachloromethane at a dose of 0.1 ml per 100 g of rat body weight in the form of a 50% oil solution. Experimental group R2 was fed a feed additive of Butaselmevit-plus for a dose of 0.1 g per 100 g of body weight together with the feed for experimental toxicosis for 30 days. On the basis of the conducted researches positive effect of feed additive "Butaselmevit-plus" on the organism of rats, which were intoxicated with tetrachloromethane, is shown, which is shown by normalization of hematological parameters and protein synthesis of liver function. Restoration of hematopoietic function of bone marrow of rats by intoxication with tetrachloromethane is due to the fact that the feed additive "Butaselmevit-plus" contains in its composition fruits thistle, which have a high level of vitamins A and K, and trace elements, namely: Ferum, Cuprum and Cobalt that are directly involved in hematopoiesis. When feeding Butaselmevit-plus as feed supplement, the number of red blood cells in the blood of the second experimental group of rats was higher than that of the first experimental group, but the control values reached only on the 30th day of the experiment. When using the feed additive Butaselmevit-plus in rats under conditions of oxidative stress development, the leukocyte count decreased to the optimum level.

Immobilization of BMP-2-derived peptides on 3D-printed porous scaffolds for enhanced osteogenesis

Three-dimensional (3D) printing technologies open up new perspectives for customizing the external shape and internal architecture of bone scaffolds. In this study, an oligopeptide (SSVPT, Ser-Ser-Val-Pro-Thr) derived from bone morphogenetic protein 2 was conjugated with a dopamine coating on a 3D-printed poly(lactic acid) (PLA) scaffold to enhance osteogenesis. Cell experiments in vitro showed that the scaffold was highly osteoconductive to the adhesion and proliferation of rat marrow mesenchymal stem cells (MSCs). In addition, RT-PCR analysis showed that the scaffold was able to promote the expression of osteogenesis-related genes, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and osteopontin (OPN). Images of the micro-CT 3D reconstruction from the rat cranial bone defect model showed that bone regeneration patterns occurred from one side edge towards the center of the area implanted with the prepared biomimetic peptide hydrogels, demonstrating significantly accelerated bone regeneration. This work will provide a basis to explore the application potential of bioactive scaffolds further.

Differentiation of bone marrow-derived mesenchymal stem cells into cardiomyocytes using different regimens of 5- azacytidine

Background: Conventional therapeutic approaches for ischemic heart disease have limited benefit in preventing post-ischemic heart failure. Therefore, stem cell therapy emerges as a hopeful tool for treatment of ischemic heart disease following myocardial infarction. Aim: To standardize a protocol ensuring the best differentiation of bone marrow mesenchymal stem cells (BM-MSCs) into cardiomyocytes by using 5-azacytidine with minimum toxicity. Materials and Methods: BM-MSCs were isolated from the bone marrow of albino rats, characterized by flow cytometry and then divided into five groups: Group I: control group, group II: exposed to differentiating medium containing 10 μmol/L of 5-azacytidine for 24 h, group III: exposed to the differentiating medium for 72 h, group IV: the differentiating medium was renewed twice weekly, group V: daily renewal of the differentiating medium was done. The experiment was terminated after 3 weeks from the induction time. Morphological changes of the cultured cells were examined under a phase contrast microscope. The effect of different duration of 5- aza exposure on BM-MSCs viability was evaluated by cell counting Kit 8 assay. Expression of cardiac troponin I and connexin 43 was measured using Quantitative reverse transcription-PCR. Transmission electron microscope was used to observe ultrastructural features of the induced cells. Morphometric study for comparing the length of the formed myofilaments in the four treated groups was done.Results: The obtained results from groups II and III showed initial limited signs of differentiation towards cardiac lineage. Groups IV and V showed optimal signs of differentiation with better results regarding group V, yet, this was at the expense of the viability of the cultured cells.Conclusion: The use of 5-aza showed a valuable differentiating effect on MSCs towards cardiomyocytes. Prolonged use resulted in promotion of the differentiation. Yet, it was at the expense of the viability of these cells.

Complementary Embryonic and Adult Cell Populations Enhance Myocardial Repair in Rat Myocardial Injury Model.

We compared the functional outcome of Isl-1+ cardiac progenitors, CD90+ bone marrow-derived progenitor cells, and the combination of the two in a rat myocardial infarction (MI) model. Isl-1+ cells were isolated from embryonic day 12.5 (E12.5) rat hearts and expanded in vitro. Thy-1+/CD90+ cells were isolated from the bone marrow of adult Sprague-Dawley rats by immunomagnetic cell sorting. Six-week-old female Sprague-Dawley rats underwent permanent left anterior descending (LAD) coronary artery ligation and received intramyocardial injection of either saline, Isl-1+ cells, CD90+ cells, or a combination of Isl-1+ and CD90+ cells, at the time of infarction. Cells were delivered transepicardially to the peri-infarct zone. Left ventricular function was assessed by transthoracic echocardiography at 1- and 4-week post-MI and by Millar catheterization (-dP/dt and +dP/dt) at 4-week post-MI. Fluorescence in situ hybridization (Isl-1+cells) and monochrystalline iron oxide nanoparticles labeling (MION; CD90+ cells) were performed to assess biodistribution of transplanted cells. Only the combination of cells demonstrated a significant improvement of cardiac function as assessed by anterior wall contractility, dP/dt (max), and dP/dt (min), compared to Isl-1+ or CD90+ cell monotherapies. In the combination cell group, viable cells were detected at week 4 when anterior wall motion was completely restored. In conclusion, the combination of Isl-1+ cardiac progenitors and adult bone marrow-derived CD90+ cells shows prolonged and robust myocardial tissue repair and provides support for the use of complementary cell populations to enhance myocardial repair.

Comparative evaluation of osteogenic activity and the effect on hematopoietic function of bone marrow of fractions of Saussurea controversa and Filipendula ulmaria extracts in experimental osteomyelitis

The aim of the work was to study the effect of fractions from Saussurea controversa and Мeadowsweet extracts on hemopoiesis and regeneration of bone tissue, which are the main target indicators in experimental osteomyelitis.Materials and methods. In the experiment we used butanol, water and element-organic fractions from the Saussurea controversa extract as well as ethyl acetate, butanol and water fractions from the Мeadowsweet extract, obtained by liquid extraction of the extracts from corresponding plants with the help of organic solvents. After modeling osteomyelitis of the right femur in rats and course treatment for 28 days, the state of bone marrow hemopoiesis was evaluated and morphological examination of the affected limb was performed.Results. The butanol fraction (at a dose of 10 mg/kg) containing flavonol glycosides, the water fraction (at a dose of 80 mg/kg) containing polysaccharides in its composition and the element-organic fraction (at a dose of 10 mg/kg) represented by organic calcium showed the highest osteogenic activity in the Saussurea controversa extract. The water fraction from the Meadowsweet extract (at a dose of 50 mg/kg) demonstrated pronounced hemopoietic activity due to stimulating erythro-, granulo- and lymphopoiesis in the bone marrow of rats with experimental osteomyelitis.

MORPHOLOGICAL PECULIARITIES OF RAT BONE MARROW UNDER THE INFLUENCE OF NITRATE SALTS IN INCREASED CONCENTRATIONS

The study of the qualitative composition of water and food products revealed the increased concentration of nitrates in their composition. Nitric acid salts in toxic doses can lead to disorders of the endocrine system, blood circulation, central nervous system, etc. Bone marrow is one of the most sensitive organs, which is responsible for the formation of cellular elements of blood. The object of our research was to study the morphological peculiarities of the bone marrow of rats under the influence of nitrate salts on the body and to determine the regularities of subacute and chronic hemotoxic effect of xenobiotics on the process of granulocyto-, erythrocyto- and thrombocytopoiesis. The study was conducted on 48 mature male rats. Nitrate Na (NaNO3) was used as xenobiotic. The animals were removed from the experiment on 10th, 30th and 90th day. The qualitative and quantitative composition of the parenchymatous and stromal component of the bone marrow was studied on the samples, stained with hematoxylin and eosin. During the experiment, the most expressed changes were observed in erythron: reduced number of nuclear cells in erythroid islands, signs of their diserythropoiesis – focal vacuolization, abnormal shape and size of the nuclei, their fragmentation, pathological inclusions in cytoplasm. Changes in granulocytopoiesis were expressed by the disturbance of their stratification – precursors with different degree of maturity were present in all parts of the medullary canal. Changes in megakaryocytes were expressed by their focal accumulation in the form of clusters and partial dislocation, regarding the sinusoids. The observations on animals revealed the term-dependent nature of changes in the bone marrow. On the 90th day, a progression of qualitative and quantitative changes in hematopoietic tissue was determined. The hemotoxic effect was expressed by disorders in the maturation of all its precursors – the ratio of parenchyma-stroma was still reduced, dysplastic changes in erythron increased, focal dystrophic changes in the stromal component of the bone marrow were observed.