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.

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.

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.

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.

Content of stromal precursor cells in heterotopic transplants of bone marrow in CBA mice of various ages.

Efficiency of colony formation of stromal precursor cells in cultured bone marrow transplants from old (24 month) CBA mice implanted to young (2-month-old) mice almost 3-fold surpassed that in cultured transplants implanted to old recipients. The content of nucleated cells in bone marrow transplants from senescence accelerated mice SAMP increased more than 2-fold, if SAMR mice with normal aging rate were used as the recipients instead of SAMP mice. Bone marrow taken from old and young CBA mice endured the same number of transplantations if the recipient mice were of the same age (5 month). It was concluded that stromal tissue considerably changes with age and is under strict control of the body.

[Proliferative and differentiation potential of individual clones derived from bone marrow stromal precursor cells]. / Proliferativnye i differentsirovochnye potentsii individual'nykh klonov stromal'nykh kletok-predshestvennikov kostnogo mozga.

We present the results of a study on the proliferative and differentiation potential of individual clones of stromal fibroblasts growing in monolayer cultures of bone marrow cells. Each precursor cell yielding a large colony in primary culture is capable of up to 34 doublings in vitro. The transplantation of clones or monoclonal strains of stromal fibroblasts into the open system results in the formation of microenvironment consisting of the bone and reticular tissue and is suitable for the differentiation of all three lines of hemopoiesis. Evidence has been obtained that, in a closed system, individual clones are capable of differentiation into the bone, cartilaginous, and reticular tissues. In other words, the adult organism has a common cell precursor for these tissues.

[Population dynamics of clonogenic stromal precursor cells in hemopoietic and lymphoid organs during bone marrow regeneration]. / Dinamika chislennosti klonogennykh stromal'nykh kletok-predshestvennikov v krovetvornykh i limfoidnykh organakh pri regeneratsii kostnogo mozga.

This paper describes our study on the regeneration of hemopoietic and stromal components of bone marrow after mechanically emptying the medullar cavity of the guinea pig tibia. The intensity of hemopoiesis was determined from the number of hemopoietic cells, while the concentration and total number of stromal precursor cells were used to estimate the ability of the bone marrow to produce stromal structures, including its ability to restore a specific microenvironment. We found that there was no direct correlation between the recovery characteristics of hemopoietic and stromal cells. An increase in the population size of stromal precursor cells takes place early after curettage, and stromal fibroblasts become phosphatase-positive according to Gomori, which is characteristic of osteogenic tissue. We have also demonstrated that curettage of 3-5 tubular bones results in the growth of this cell population in the bone marrow of nonoperated bones and even in the spleen, which in guinea pigs participates only in lymphopoiesis.

[Quantitative characteristics of the transfer of the hematopoietic microenvironment]. / Kolichestvennye kharakteristiki perenosa gemopoéticheskogo mikrookruzheniia.

Heterotopic transplantation of marrow fragments results in the formation of new bone marrow organs. The amount of hemopoietic cells populating these organs is connected nonlinearly with the volume of the grafted tissue or with the number of transplanted marrow cells. Statistical analysis points out that the number of the populating cells depends on the radius of the initial bone marrow transplant. Thus, the previous data on the radiosensitivity of the microenvironmental transfering stromal cells and on their concentration obtained by measuring the size of the heterotopic bone marrow organs have turned out incorrect.

[Effect of the stem cell inhibitory factor on the formation of hemopoietic colonies]. / Vliianie faktora ingibitsii stvolovykh kletok na formirovanie gemopoéticheskikh kolonii.

The activated T lymphocytes release in the culture medium a factor inhibiting the ability of hemopoietic stem cells to form hemopoietic colonies in the spleens of lethally irradiated recipients. An analysis of the effect of this factor on hemopoietic colonies was carried out. Its inhibiting effect was not related to the process of opsonization and to changes in the migration of spleen cells in the irradiated organism. No marked changes in the ratio of erythroid, myeloid and megakaryocytic foci were found in the spleens of irradiated mice which received the syngeneic bone marrow cells treated in vitro by the factor of stem cell inhibition: all three sources of hemopoiesis suffered under its effect to the same extent and their total number of the spleens was sharply reduced. Some mechanisms of the effect of the factor of stem cell inhibition on the formation of hemopoietic foci are discussed.

[Formation of hematopoietic territories in heterotopic bone marrow transplantation]. / Obrazovanie krovetvornykh territorii pri geterotopnoi peresadke kostnogo mozga.

The possibility of the formation of a new bone marrow organ from the initially isolated bone marrow cells has been proved for the first time. Suspensions of the bone marrow cells (5 x 10(6)) placed on millipore filters were grafted under the renal capsule in mice. As a result, the bone formed and the medullary cavity developed which was populated by hemopo etc cells. These processes were closely followed during 9 weeks. To compare the sizes of hemopoietic territories formed by grafting of different bone marrow fragments, the number of bone marrow cells in the heterotopic bone marrow was estimated. The bone marrow organs formed under the renal capsule after grafting bone marrow from the whole femur or its half contained practically the same number of hemopoietic cells. It was also true for the subcutaneous transplants of bone cylinders of the same size but with different amount of bone marrow. Hence it follows that the amount of hemopoietic territory grafted depends to a great extent on the transplant form and, therefore, does not reflect the number of initially grafted microenvironment-forming cells.

Origin of bone marrow stromal mechanocytes in radiochimeras and heterotopic transplants.

Strain histocompatibility antigens (detected by indirect immunofluorescence reaction) and sex chromosomes were used as markers of donor and recipient cells in monolayer cultures of bone marrow from radiochimeras and from heterotopic transplants. In contrast to macrophages and hemopoietic cells all bone marrow fibroblasts precursors in radiochimeras were of recipient origin; in heterotopic transplants they were of donor origin. These data point to the decisive role of stromal mechanocytes, and not macrophages or hemopoietic cells, in creating the bone marrow hematopoietic microenvironment.

[Origin of stromal bone marrow mechanocytes according to the results of typing them by isoantigens and chromosomal markers]. / Proiskhozhdenie stromal'nykh mekhanotsitov kostnogo mozga po dannym ikh tipirovaniia po izoantigenam i khromosomnym markeram.

The bone marrow of radiochimaeras and heterotopic bone marrow transplants were used to study the origin of precursors of the fibroblasts growing in the monolayer cultures of hemopoietic tissue. In the bone marrow explants of the (C57BL/6 X CBA) F1 mice, in which the CBA bone marrow was transplanted following the lethal irradiation, the fibroblasts grown in the colonies were of recipient origin judging by isoantigens in the reaction of indirect immunofluorescence with the anti-C57BL/6-serum. At the same time in the bone marrow explants from heterotopic transplants (CBA leads to CBA X C57BL/6) the fibroblasts grown in colonies were of donor origin. The cultures of hemopoietic cells of the bone marrow of females heterotopically transplanted in the singenic male (guinea pigs Huston) contained only fibroblasts which were of donor origin judging by sex chromosomes in the metaphase plates of dividing cells. Hence, the bone marrow precursors of fibroblasts do not depend histogenetically on hemopoietic cells and are not replaced at the expense of repopulating cells of the second partner.

[Development of hematopietic and lymphoid tissues in conjoint bone marrow and thymus transplants]. / Razvitie krovetvornoi i limfoidnoi tkanei v sovmestnykh transplantatakh kostnogo mozga i timusa.

The model of heterotopic transplantation of the mixture of bone marrow and thymus fragments was used to study the interaction of hemopoietic and lymphoid tissues under their direct contact. The bone marrow and thymus fragments of adult mice F1 (CBAXXC57BL) were transplanted separately or in the mixture under the kidney capsule of mice of the same strain. During the whole period of observation (from 10 days up to 14 months), the development of bone marrow and thymus fragments in the joint transplants proceeded independently, no "mixed" stroma appeared, and the stroma of each organ ensured the differentiation characteristic of its organ. The development of joint transplants somewhat differs from that of isolated transplants: on the 10th day a greater amount of hemopoietic tissues was noted in the former; the bone marrow component increases continuously up to 6 months (vs. 1--2 months in the isolated transplants); the bone and hemopoietic tissues predominate in the joint transplants by 14 months, the amount of thymic tissue markedly decreases but it does not disappear completely.

[Origin of stromal mechanocytes in the bone marrow cell cultures]. / Proiskhozhdenie stromal'nykh mekhanotsitov v kul'turakh kostnogo.

The method of indirect immunofluorescence with the use of the isolinear antiserum was applied to the study of the linear reference of fibroblasts growing in the monolayer bone marrow cultures from the semisyngenous heterotropic transplants and radiochimerae. As shown, fibroblasts from the bone marrow of complete radiochimerae were of the recipient origin, whereas the histiocytes-macrophages (in the same cultures) - of donor origin. All the fibroblasts in the bone marrow cultures of the heterotropic transplant belonged to the initial donor, whereas 80% of the histiocytes were recipent's. This is a direct proof of the fact that stromal mechanocytes and hemopoietic cells, and also macrophages belonged to different cell lins.

[Effect of various factors on the differentiation of hematopoietic stem cells]. / Vliianie nekotorykh faktorov na differentsirovku stvolovykh gemopoeticheskikh kletok

The treatment in vitro of bone marrow cells in mice by phytohemagglutinin, concanavaline, or antilimphocytic globulin resulted in the suppression of exogenous hemopoietic colonies in the spleen of lethally irradiated (830r) syngenic recipients, whereas lipopolysaccharide, tuberculin, anti-theta serum or nati-gamma-globulin serum exerted no influence on the colony-forming function of hemopoietic stem cells. The morphological analysis of the ratio and cell composition of hemopoietic colonies has revealed no marked differences between the experimental and control groups. The suppression of hemopoietic stem cells by mitogens might be due both to their direct effect and indirect one, possibly, through a humoral factor.

[Restoration of the osteogenic properties of bone marrow following irradiation according to the findings of heterotopic transplantation]. / O vosstanovlenii osteogennykh svoistv kostnogo mozga posle oblucheniia po dannym geterotapnoi transplantatsii

It was found that bone marrow obtained from the tibium or the femur (locally irradiated with 2000 R) and transplanted subcapsularly into the kidney of mice of the same strain 0-4 months after the irradiation was always resorpted. As to the bone marrow from the nonirradiated bones of the same donors--it always possessed osteogenic potencies. Stroma of the irradiated limb was defective ang failed to regenerate at the expense of repopulation of the stroma elements from the nonirradiated sections. Analogous conclusions were drawn on the basis of the experimental results with repeated irradiation of mice with the shielded extremity. In difference to the hemopoietic, stromal elements were incapable of repopulation (according to the data of heterotopic transplantation).