Laser Thermo-Photobiomodulation of Bone Marrow Mesenchymal Stem Cells.

We studied the effect of laser radiation of moderate intensity with a wavelength of 970 nm on the efficiency of colony formation of rat bone marrow mesenchymal stem cells (MSC) in vitro. In this case, photobimodulation and thermal heating of MSC occur simultaneously. This combined laser treatment allows increasing the number of colonies by 6 times in comparison with the control and by more than 3 times in comparison with thermal heating alone. The mechanism of such an increase is associated with combined thermal and light effects of laser radiation of moderate intensity, which stimulates cell proliferation. This phenomenon can be used as the basis for solving the most important task of cell transplantation, associated with the expansion of autologous stem cells and activation of their proliferative potential.

Changes in the Number of Mesenchymal Stem Cells in Bone Marrow at Different Periods after In Vivo Exposure of the Bone Marrow to Local Infrared Laser Radiation.

We determined optimal parameters of bone marrow (BM) irradiation in vivo for rapid increase in the number of mesenchymal stem cells (MSC) at the initial stages of the culturing without changing the karyotype, polyploidy, which are observed at higher passages. Such an increase is necessary to achieve the required number of cells at the initial passages for subsequent transplantation into the body. It was shown that after irradiation with λ=0.97 µm, the maximum and similar increase in the content of BM MSC in comparison with the control (by 2.4 times) was observed on day 2 in the irradiated and contralateral tibia. An insignificant difference in the number of BM MSC for the irradiated and contralateral tibia remained at all terms after irradiation, with a general decrease in the number of BM MSC up to 21 days. After laser irradiation with λ=1.56 µm, the maximum number of BM MSC was also observed on day 2. In this case, the concentration of these cells in the irradiated and contralateral limbs exceeded the control by 3.1 and 1.7 times, respectively. With increasing the time after exposure, the number of BM MSC in both limbs showed the same tendency to a decrease as after irradiation at λ=0.97 µm.

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.

[Colony-forming fibroblast precursors of the circulating blood]. / Kolonieobrazuiushchie predshestvenniki fibroblastov tsirkuliruiushchei krovi.

Colony-forming fibroblast precursors were detected in circulating blood of adult guinea pigs by CFUf in vitro colony assay. SFU amount to 0.9 +/- 0.2 (M +/- m) per 10(5) explanted leucocytes ranging from 0.04 X 10(-5) to 3.0 X 10(-5) for individual donors. The presence of collagen type I and lack of factor VIII antigen and of FC-receptors proved that CFU-derived colonies in the blood cultures were composed of fibroblasts.

[Osteogenic properties of adhesive cells in Dexter culture of the mouse bone marrow]. / Osteogennye svoistva adgezivnykh kletok iz deksterovskikh kul'tur kostnogo mozga myshi.

Disaggregated cell suspensions obtained by mouse bone marrow fermentative digestion as well as stromal tissue obtained by marrow mild mechanical destruction were explanted. Both methods yield the cultures in which the hematopoiesis duration is comparable with dexter cultures. Adhesive cells from all of these three culture types were resuspended and in the porous gelatin sponges heterotopically transplanted under the kidney capsule of syngenic recipients. In the transplantation site there develops the hemopoietic organ containing reticular stroma, hemopoietic cells, and in most cases the well developed bone tissue. Thus, the adherent layers of mouse bone marrow dexter and similar cultures contain for a long period (not less than 2-3.5 months) the stromal fibroblast population which maintains its osteogenic and hemopoietic microenvironment transfer capacities.

[Effect of bone marrow cells on colony-forming stromal cells in guinea pigs and on the proliferation of their cultured descendents]. / Vliianie kostnomozgovykh kletok na koloneobrazuiushchie stromal'nye kletki morskikh svinok i proliferatsiiu ikh kul'tural'nykh potomkov.

In the presence of irradiated bone marrow cells the efficiency of stromal colony formation increases from 0.8 +/- 0.2 to 3.6 +/- 0.4 per 10(4) explanted bone marrow cells. The growth-stimulating activity of bone marrow cells on passaged bone marrow fibroblasts depends on growth conditions of passages to which irradiated bone marrow cells are added. The response of proliferating bone marrow fibroblasts to stimulating activity of bone marrow cells is low, while addition of bone marrow cells to fibroblast cultures stimulates their proliferation.

[Clonal nature of fibroblast colonies formed by stromal bone marrow cells in culture]. / Klonal'naia priroda kolonii fibroblastov, obrazuemykh stromal'nymi kostnomozgovymi kletkami v kul'turakh.

The clonal nature of CFUf-derived fibroblast colonies was tested in mixed cultures of CBA and CBAT6T6 bone marrow cells. Inoculation of marrow cell suspensions into flasks coated with poly-I-lysin has proved that no stromal aggregates were present among cells subjected to explantation. Marrow cell cultures depleted of macrophages and myeloid cells were used for chromosome analysis. The coincidence of karyotypes within a stromal colony was found in mixed cultures, which proves that CFUf-derived fibroblast colonies are cell clones.

[The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form]. / Soderzhanie stromal'nykh kolonieobrazuiushchikh kletok (KOKf) v kostnom mozge myshei i klonal'naia priroda obrazuemykh imi kolonii fibroblastov.

The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells. Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells. The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones. It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts. The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow). In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.

Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges.

Cells for study were obtained from mouse bone marrow by three methods: 1) Mechanical dissociation; 2) Trypsin release; and 3) Passage through a syringe and needle. The ratio of the fibroblast colony-forming cells in these suspensions was, respectively, 1:13:1. Of the colonies formed by cells obtained by trypsin release, 70% were alkaline phosphatase positive fibroblasts. Freshly isolated marrow cells and cells harvested, by trypsin treatment, from cultures established from the differently obtained bone marrow cell suspensions, were absorbed into porous sponges for transplantation under the renal capsule of mice. Ossicles containing bone marrow formed after transplantation of either freshly isolated cells, or cultured cells obtained from marrow by trypsin treatment, but not after the transplantation of cells cultured from marrow obtained by mechanical dissociation. The size of the bone marrow "organs" that formed was determined by both the number of cells grafted and the size of the sponge in which they had been absorbed for grafting.

[Formation of bone marrow organs following transplantation of cell suspensions to sponges]. / Obrazovanie kostnomozgovykh organov pri transplantatsii kletochnykh suspenzii v poristykh gubkakh.

Heterotopic transplantation of bone marrow monocellular suspensions in gelatin and collagenous sponges is highly effective in promoting osteal tissue formation and organization of hemopoietic microenvironment. This depends on the presence in the transplant of a framework for the growth of stromal mechanocytes. Formation of bone marrow organs proceeds at a greater efficacy on using trypsinized bone marrow cells versus those isolated by mechanical techniques.

[Transfer of bone marrow microenvironment by stromal cells grown in culture and transplanted into sponges]. / Perenos kostnomozgovogo mikrookruzheniia stromal'nymi kletkami, vyrashchennymi v kul'turakh i transplantirovannymi v zhelatinovykh gubkakh.

Monocellular suspensions from trypsinized CBA mouse bone marrow cells were explanted to monolayer cultures. The cells of the adhesive layer withdrawn from 15-30-day cultures were transplanted in gelatin sponges beneath the kidney capsule from syngeneic recipients. The transplantation was accompanied by bone formation and by transfer of bone marrow microenvironment in the overwhelming majority of cases.

[Heterologous antisera to stromal mechanocytes of the bone marrow]. / Geterologicheskie antisyvorotki k stromal'nym mekhanotsitam kostnogo mozga.

Heterologous rabbit antifibroblast serum (AFS) to stromal fibroblasts of the guinea-pig bone marrow was obtained. AFS was shown to bind specifically stromal fibroblasts and their precursors (but not macrophages) in the monolayer primary cultures of the bone marrow, thymus and spleen of guinea pigs (in the complement-dependent cytotoxic reaction and in the indirect immunofluorescence test); AFS also bound precursors of blood fibroblasts and peritoneal exudate (in the cytotoxic reaction). Precursors of the thymus fibroblasts were found to be more sensitive to the AFS action than the spleen and bone marrow precursors, this suggesting that the thymic stromal mechanocytes had a greater concentration of common tissue-specific antigens on their surface. Precursors of the stromal fibroblasts in native cell suspensions were found to be essentially more sensitive to the cytotoxic action of AFS than the colony-forming fibroblasts on the passage cultures.