Comparative Morphological Study of the Formation of Reparative Regenerate during Skin Wound Healing in Rats under the Effect of Drugs and Bone Marrow.

We studied the formation of the reparative regenerate of the skin wound in rats under the effect of drug products based on keratan and secretome of bone marrow mesenchymal stem cells (MSC), as well as bone marrow cells (native and exposed to laser radiation with a wavelength of 1.56 µm). Due to the biological affinity for the dermal tissue, keratan preparations being applied to the skin stimulate regeneration of the wound defect. This substance in the form of a gel is characterized by high diffusion capacity, penetrates into the deeper layers of the dermis, and promotes the growth of the granulation tissue. Application of an ointment prepared on the basis of MSC secretome promotes quick transition of the healing process from the inflammatory to the regenerative stage. Thus, bone marrow cells were successfully used for skin wound healing. The results of the use of bone marrow cells for the healing of skin wounds were successful; bone marrow exposed to laser radiation demonstrated high efficiency in promoting reparative processes.

Effect of Non-Thermal Plasma on Proliferative Activity and Adhesion of Multipotent Stromal Cells to Scaffolds Developed for Tissue-Engineered Constructs.

We studied the effect of non-thermal argon plasma on proliferative activity of bone marrow multipotent stromal cells in vitro. Treatment of stromal cell suspension with pure argon did not affect their proliferation. The cells treated with non-thermal argon plasma and explanted in the treatment medium demonstrated growth inhibition by 30-40% in comparison with the control. Multipotent stromal cells treated with plasma and after centrifugation explanted in normal medium within 12 min demonstrated accelerated growth. The total cell growth from the pellet and supernatant significantly exceeded the control values. We also analyzed adhesion and proliferative activity of multipotent stromal cells treated with non-thermal plasma on bioresorbable carriers. The cells adhered and proliferated on all types of studied samples. Adhesion properties of scaffolds differed. Caprolactone was found to be the most suitable material for adhesion and proliferation of multipotent stromal cells.

Reinforced Hybrid Collagen Sponges for Tissue Engineering.

We created an anisotropic material based on collagen sponge and reactive polylactide structured by laser photopolymerization. The combination of collagen with reactive polylactide improves the resistance of the formed matrices to biodegradation in comparison with collagen sponge, while the existence of sites with different mechanical characteristics and cell affinity on the matrix provides directed cell growth during their culturing. It was shown that reinforcement of the collagen sponges 7-fold increased the mean Young's modulus for the hybrid matrix without affecting its cytotoxicity. The developed matrix provides cell adhesion and proliferation along reinforcement lines and can be used for fabrication of tissue engineering constructs.

Reconstruction of Ligament and Tendon Defects Using Cell Technologies.

We studied the possibility of restoring the integrity of the Achilles tendon in rabbits using autologous multipotent stromal cells. Collagen or gelatin sponges populated with cells were placed in a resorbable Vicryl mesh tube and this tissue-engineered construct was introduced into a defect of the middle part of the Achilles tendon. In 4 months, histological analysis showed complete regeneration of the tendon with the formation of parallel collagen fibers, spindle-shaped tenocytes, and newly formed vessels.

Effects of acoustic and EHF impulses on multipotent stromal cells during formation of bone marrow containing heterotopic organs in tissue engineered constructions.

We studied the effects of physical factors (acoustic impulses of laser-induced hydrodynamics, AILIH, and EHF-radiation) on the formation of heterotopic bone marrow organs. Suspension of precipitated mouse bone marrow cells was exposed to AILIH and EHF or their combinations (AILIH+EHF, EHF+AILIH). The developed tissue engineering constructions (gelatin sponges containing 107 nucleated bone marrow cells exposed to physical factors) were transplanted under the renal capsule of syngeneic mice. Analysis of newly formed hemopoietic organs was performed after 3 and 5 months. The total amount of hemopoietic cells, number of multipotent stromal cells, efficiency of colony formation from these cells, and weight of bone capsule of the transplants were measured. Microscopic study showed that 5-month transplants were significantly larger than 3-month transplants and contained 3-fold more hemopoietic cells (20-fold in the AILIH+EHF group). The number of multipotent stromal cells was maximum in EHF+AILIH group (by 2.2 times higher than in the control) and minimum in AILIH+EHF group. Exposure to EHF+AILIH had most pronounced effect on the formation of the bone marrow transplants. The weight of bone capsules more rapidly increased in gelatin sponges of 3-month transplants of EHF+AILIH and AILIH groups. These data suggest that the studied physical factors can be used for acceleration of rehabilitation process.

Comparison of the efficiency of transplantation of bone marrow multipotent mesenchymal stromal cells cultured under normoxic and hypoxic conditions and their conditioned media on the model of acute lung injury.

The therapeutic efficiency of intravenous injection of rat bone marrow multipotent mesenchymal stromal cells grown under conditions of normoxia and hypoxia (3% O2) and conditioned media from these cultures were compared on the rat model of acute lung injury induced by intraperitoneal injection of lipopolysaccharide. The best therapeutic efficiency was demonstrated by cells grown under hypoxic conditions. The effect of conditioned media was less pronounced and did not depend on the culturing conditions.

Effect of BMP-2 protein on the count and osteogenic properties of multipotent stromal cells and expression of cytokine genes in primary cultures of bone marrow and spleen cells from CBA mice immunized with bacterial antigens.

We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines.

Proline-rich hypothalamic polypeptide has opposite effects on the proliferation of human normal bone marrow stromal cells and human giant-cell tumour stromal cells.

In the present study we carried out experiments in vitro and in vivo and investigated the effect of proline-rich polypeptide (PRP) on the proliferation and effectiveness of colony formation of MMSCs in vitro. Various routes and doses of PRP administration to rats increased the number of MMSCs in bone marrow and spleen. Our research revealed opposite effects of PRP on the proliferation of bone marrow stromal cells obtained from normal humans and stromal cells isolated from a human giant-cell tumour.

Effect of alpha-fetoprotein on the count of bone marrow and splenic stromal precursor cells and proliferation of their cultural descendants.

The efficiency of cloning of stromal precursor cell increased more than 2-fold in splenic cultures and more than 3-fold in bone marrow cultures 24 h after injection of Profetal preparation to mice in vivo. The number of nucleated cells did not change in the bone marrow and slightly increased in the spleen. Addition of Profetal in vitro 2-fold decreased the efficiency of stromal precursor cells colony formation in mouse splenic cultures and dose-dependently decreased this process in bone marrow cultures derived from these animals, the maximum (5-fold) inhibitory effect was observed in a dose of 50 microg/ml. Addition of Profetal to cultured human bone marrow fibroblasts did not change the content of stromal fibroblasts in cultures. These data indicate the possibility of indirect effect of alpha-fetoprotein on the number of stromal precursor cell in hemopoietic and lymphoid organs.

The effect of epinephrine on feeding and motion patterns in goldfish Carassius auratus (L.).

The effect of intraperitoneal injections of epinephrine (0.14 and 0.70 mg/kg) on some characteristics of feeding activity (ration and total time of feeding) as well as on motion patterns (time of swimming in group and separately) in juvenile goldfish has been investigated. Two-phase (short-term decrease in the first phase, increase in the second one) feeding response under both doses of epinephrine has been revealed. More pronounced effect of epinephrine at the dose of 0.14 mg/kg on the ration and time of feeding (comparing to the dose of 0.70 mg/kg and Ringer injection) was observed in the second phase. Furthermore, significant decrease of time of "separated" swimming in the first phase under both doses of the hormone is revealed. The hyperglycemic response induced by the injections of epinephrine, with synchronous reduction of the concentration of glycogen in hepatopancreas allows to suggest that glycogen-phosphorylase activating mechanism was underlying the "first-phase" change of feeding reactions of goldfish.

Characteristics of human bone marrow mesenchymal stem cells isolated by immunomagnetic selection.

Immunophenotype of human bone marrow mesenchymal stem cells was studied after several culturing passages and after cryopreservation. Immunocytochemical analysis showed that bone marrow mesenchymal stem cells acquired homogeneity during in vitro culturing, but initially contained heterogeneous populations.

Comparative analysis of differentiation and behavior of human neural and mesenchymal stem cells in vitro and in vivo.

Comparative analysis of differentiation of human neural and mesenchymal stem cells in tissue culture and after transplantation into the brain was carried out using the same antibody set. Neural stem cells differentiated into all types of neural cells, are retained after transplantation, migrate, and form reciprocal relationships with the recipient brain. Mesenchymal stem cells were incapable of neural development under conditions of common culturing or after transplantation and retained the fibroblast-like status. Recipient filaments grew into mesenchymal stem cell transplants containing no neural cells due to local changes in the extracellular matrix at the site of transplantation.