The dynamics of proliferation and differentiation of osteogenic cells under supportive unloading.

With the use of radioactive marker of DNA synthesis--3H-thymidine we have studied the dynamics, peculiarities of proliferation and differentiation of osteogenic cells under hind limb unloading of white rats ("tail suspension" method at an angle 35 degrees) during 28 days. The 3H-thymidine was administered at a single dose at the end of the experiment, the biosamples were taken from femoral bones in 1, 48, 96 hr. Light and electron-microscopic radioautography with 3H-thymidine (in 1 hour) have shown, that basic fraction of DNA synthesizing cells in the zones of adaptive remodelling of bone tissue is represented by little-differentiated perivascular cells (that include osteogenic cell precursors). A tendency for a decrease of a labelling index in the 3H-thymidine osteogenic cells on metaphyseal bone trabeculae under hind limb unloading has been established. The dynamics of labelled cells during various time intervals after 3H-thymidine injection testifies to a delay in the differentiation precursors in osteoblasts and their transformation to osteocytes in experiment animals. The obtained data have shown that a long-term supportive unloading leads to lowering the intensity of osteogenetic processes in long bones and reducing bone mass.

Interactions of cells in zones of bone resorption under microgravity and hypokinesia.

With the use of the methods of electron microscopy and autoradiography employing 3H-glycine the study was made of some morpho-functional cells-cells interactions (osteoblasts, osteocytes, macrophages, fibroblasts) in zones of adaptive remodeling of bone structures of the metaepiphyseal femoral bones of white rats which were during 28 days under experimental hypokinesia conditions, as well as of rats, flown on SLS-2 during 2 weeks. It is established that in zones of an increase of mineral matrix resorption some osteoblasts and osteocytes undergo destruction; a part of osteoblasts remains intact. The osteoclasts don't take part in destruction of osteoblasts and osteocytes. The utilization of the osteogenic cells detritus is accomplished by macrophages, coming to these zones. The resorption loci are filled not with the differentiating osteoblastic cells, as it is the case in the norm, but with fibroblasts and the bundles of collagen fibrils (fibrotic tissue) which do not undergo mineralization. Such changes are considered as one of the mechanisms of bone tissue response to a reduction of the supporting load.

Changes of cell-vascular complex in zones of adaptive remodeling of the bone tissue under microgravity conditions.

We examined the peculiarities of the structure of the blood-vascular bed and perivascular cells in zones of osteogenesis in the epiphyses and metaphises of femoral bones of rats, flown aboard the US laboratory SLS-2 for two weeks by electron microscopy and histochemistry. In zones of bone remodeling, there was a tendency for a reduction of sinusoid capillary specific volume. Endotheliocytes preserve the typical structure. In the population of perivascular cells, we discovered differentiating osteogenic cells that contained alkaline phosphomonoesterase as well as cells that don't contain this enzyme and differentiate into fibroblasts. The fibroblasts genesis in zones of adaptive remodeling of spongy bones leads to a further development of fibrous tissue that is not subject to mineralization.

Mechanisms of gravity-dependent changes in the bone tissue.

The most typical changes for the bone under the space flight conditions and a long-term hypokinesia are the following: the decreasing in bone mass, the demineralization and a reducing of a mechanical strength. It can lead to osteopenia and osteoporosis development. Also it increases the risk of fractures of supporting bones. Osteopenies, caused by the microgravity, are partially connected with the increasing of a reduction of trabecular bones. [Cytological mechanisms of gravity-dependent reactions in a bone tissue remain in many respects not clear. The study purpose was the analysis of some ultrastructural changes in bone tissue cells of the monkeys (Macaca mulatta), staying during 2 weeks onboard the biosatellite "Bion-11".

Ultrastructural changes in osteocytes in microgravity conditions.

We examined the histology and morphometry of biosamples (biopsies) of the iliac crest of monkeys, flown 14 days aboard the "Bion-11", using electron microscopy. We found, that some young osteocytes take part in the activation of collagen protein biosynthesis in the adaptive remodeling process of the bone tissue to microgravity conditions. Osteocyte lacunae filled with collagen fibrils; this correlates with fibrotic osteoblast reorganization in such zones. The osteolytic activity in mature osteocytes is intensified. As a result of osteocyte destruction, the quantity of empty osteocytic lacunae in the bone tissue increases.

Morpho-functional adaptations in the bone tissue under the space flight conditions.

Microgravity in space flight--situation of a maximum deficit of supporting loading on the skeleton and good model for finding-out of osteopenia and osteoporosis development laws, which are wide-spreading now and are "civilization diseases". Most typical for bones in conditions of a microgravitation by changes are: a decrease of intensity growth and osteoplastic processes, osteopenia and osteoporosis, decreasing of a mechanical strength and the risk of breaches arising (Oganov V.S., Schneider V. (1996)). Cytological mechanisms of gravity-dependent reactions in a bone tissue remain in many respects not-clear. By the purpose of our work was the analysis of some ultrastructural changes in bone tissue cells of the monkeys (Macaca mulatta), staying during two weeks onboard the biosatellite BION -11.

Bone ultrastructural changes in Bion 11 rhesus monkeys.

Iliac crest biopsies of Bion 11 monkeys were examined by electron microscopy. The flight samples contained a large number of inactive osteoblasts. Together with ultrastructural changes of the rough endoplasmic reticulum and the Golgi complex, the predominance of inactive osteoblasts pointed to a lower rate of specific syntheses. Some osteoblasts were transformed to fibroblast-like cells. It was found that osteogenesis declined, osteoid mineralization changed, and fibrotic zones developed.

[The effect of microgravity on osteogenic cells in culture. Ultrastructural research]. / Vliianie mikrogravitatsii na osteogennye kletki v kul'ture. Ul'trastrukturnoe issledovanie.

In order to estimate the influence of microgravity on osteogenic cells a comparative ultrastructural study of preosteoblastic cells of line MN7, cultivated in space and in ground conditions has been carried out. Morphometric analysis has revealed that in cells exposed during 9 days on board the biosatellite Cosmos 2229 endoplasmic reticulum was not so expensive and the number of myeline-like structures increased as compared to the ground control. Percentage of cells with nucleoli is lower in the flight samples as well. The increase in specific volume of lipid droplets and changed structure of mitochondria testifies to deviation in energy metabolism. The reaction of osteogenic cells to microgravity resulting in suppression of specific functions may contribute to alterations in the bone metabolism under the space flight conditions.

Changes in the numbers of osteoclasts in newts under conditions of microgravity.

Intensity of osteoclastic resorption and calcium content were investigated in intact limb bones of the newts flown on board of a biosatellite Cosmos-2229 after amputation of their forelimbs and tail. Using X-ray microanalysis it was shown an increase in calcium content in the bones on 20th day after operation. Histological study revealed an activation of osteoclastic resorption on endosteal surface of long bones. The newts exposed after surgery on a biosatellite had the same level of bone mineralisation as operated ground control ones, but the increase in number of polynuclear osteoclasts was lower.

[The role of osteoclasts in maintaining calcium homeostasis in newts]. / Rol' osteoklastov v podderzhanii gomeostaza kal'tsiia u tritonov.

The intensity of osteoclastic resorption and calcium contact were investigated in intact limb bones of the newts flown onboard of biosatellite Cosmos-2229 after amputation of their forelimbs and tail. X-ray microanalysis has shown an increase of calcium content in the bones on the 20th day after operation. Histological study revealed activated osteoclastic resorption on the inner surface of long bones. The newts exposed to weightlessness after the operation had the same level of bone mineralization as the operated ground control ones, but the number of polynuclear osteoclasts increased to a lesser extent.

[Osteoblasts during various functional states]. / Osteoblasty pri razlichnykh funktsional'nykh sostoianiakh.

At the electron microscopical investigation of osteoblasts in different zones of osteogenesis (enchondral foci, metaphyses, endosteum) in the rat and rabbit femoral bone it has been revealed that their population is heteromorphic. As demonstrate cytochemical data and radioautography, using 3H-uridine, 3H-glycine, 35S-sulfate, 45Ca, results of measurements in osteoblast population, 4 morpho-functional states (or types) are defined. In areas of an intensive osteoplastic process there are young osteoblasts (I type), mature functionally active osteoblasts (II type), osteoblasts with a hypertrophic endoplasmic network (cell-depots of the secrete--III type). In preosteoblasts and osteoblasts of the I type a higher than in osteoblasts of other types intensity of 35S-sulfate incorporation and alkaline phosphatase activity is revealed. In osteoblasts of the II type processes of biosynthesis of collagenous proteins predominate. Osteoblasts of the III type are subjected to destruction during secretion process. In the areas where osteopoesis is dying away, osteoblasts of the I and II types transform into a poorly active state, concerning specific biosynthesis (osteoblasts of the IV type). Presence of osteoblasts having various functional states in the areas of intensive osteopoiesis, depends on certain asynchronity of specific processes of biosyntheses, that occur in them. The morpho-functional states described are regarded as a specific peculiarity in function of collagene-producing cells.

[Reproduction and differentiation of the cells in enchondral osteogenesis]. / Reproduktsiia i differentsirovka kletok v énkhondral'nom osteogeneze.

Light and electron-microscopical 3H-thymidine autoradiography was used to study the dynamics of cell populations in the zones of enchondral osteogenesis in a tubular bone. In the early postnatal ontogenesis little differentiated perivascular cells are characterized by the highest proliferative activity in this region; they are considered as a population containing initial forms of the histogenetic sequence (differon) of the stromal fibroblast-like cells including osteoblasts. Differentiation of osteogenic cells from the initial forms to the mature osteoblasts proceeds through a number of successive divisions (1-3 divisions) and is accompanied by a decrease in the proliferative activity due to the increase in the generation time and decrease in the cell proliferative pool. The major part of osteoblasts is outside the mitotic cycle. At the later stages of ontogenesis the intensity of growth processes in the bone is provided for by changes in the proliferative pool of the committed precursor cells (preosteoblasts) which make a part of endosteum, vascular channels and bone marrow stroma.

[Chondroclastic properties of the cartilaginous tissue macrophages of developing bone]. / Khondroklasticheskie svoistva makrofagov khriashchevoi tkani razvivaiushcheisia kosti.

By the electron microscopic and cytochemical studies, in the vascular canals of cartilaginous epiphyses and in the zones of endochondral process of the rabbit and rat femoral bones, there were revealed monocytes, macrophages, the cells of the intermediate differentiation stages among the perivascularly located forms. In the lysosomes as well as on the surface of cytoplasmic membrane of mature macrophages the activity of acid phosphatase and non-specific esterase was demonstrable. Phagolysosomes showed accumulation of 35S-sulphate label 2 hours after its injection into an organism. Macrophages cause degradation of non-mineralized cartilaginous matrix by secreting hydrolases and other enzymes and then phagocytize the preformed substrate. In the zones of endochondral process, macrophages also penetrate into the "capsules" of hypertrophic chondrocytes and are involved in their destruction. The cells are regarded as tissue--specific macrophages--chondroclasts.

[Stromal cells of the bone marrow in growing bone]. / Kletki stromy kostnogo mozga v rastushchei kosti.

By means of electron microscopy, cytochemistry and radioautography with 3H-thymidine, the bone marrow stromal cells have been studied in the zones of endochondral osteogenesis in the rabbit and rat femoral bones. In the stromal cells demonstrating a high alkaline phosphatase activity are distinguished: perivascular, reticular fibroblastic, osteogenic cells. Populations of the perivascular phosphatase-positive cells include poorly differentiated DNA-synthesizing forms, as well as cells with signs of differentiation into stromal fibroblasts. Cleft-like spaces in cytoplasm of the fibroblastic reticular cells are, probably, formed as a result of lymphocyte-like mononuclears passing through. Phagocyting stromal elements are presented by macrophages, having perivascular localization and including into composition of erythroblastic islets. Mononuclear macrophages are revealed also on the surface of osseous trabecules, where they participate in destruction of hemopoetic and osteogenic cells.

[Submicroscopic changes in the capillary cells of an area of osteogenesis during body uptake of lead acetate]. / Submikroskopicheskie izmeneniia v kletkakh kapilliarov zony osteogeneza pri postuplenii v organizm atsetata svintsa.

Ultrastructurometry has revealed that daily entry of lead acetate in a dose of 50-100 mg/kg per body weight for 1-2.5 months increases chromatin condensation in nuclei, promotes swelling and myelin-like degeneration of the mitochondria, hyperplasia of the endoplasmic net, as well as appearance of the "enlightment" zones in the cytoplasm of endotheliocytes and perivascular cells of the capillaries from a metaphyseal zone of the rat femur. These changes are a consequent of the edema of cells and of the decrease in their biosynthetic activity.

[Dynamics and ultrastructural characteristics of osteoclast formation]. / Dinamika i ul'trastrukturnye osobennosti obrazovaniia osteoklastov.

Using electron microscopy and 3H-thymidine autoradiography it has been shown that osteoclasts of rats and rabbits are formed by fusion of cell precursors which become members of the osteoclasts 14 hours following S-phase. Monoblasts and promonocytes are considered as DNA-synthesizing forms. A single injection of parathormone in a dose of 10 units/100 g of body weight leads to an increase in the fractions of DNA-synthesizing cells. Monocytes and macrophages appear to be direct osteoclast precursors in resorption zones. As a result of their fusion there appear "unfixed" polynuclear macrophages (young osteoclasts). The number of osteoclast nuclei increases also at the expense of little-differentiated phagocytes, the inclusion of the latter takes place at different stages of the life span of osteoclasts depending on the intensity of bone resorption processes. The structural characteristics of fusion of cell precursors are considered.

[Ultrastructure of DNA-synthesizing cells in an enchondral focus and osteogenic precursor cells]. / Ul'trastruktura DNK-sinteziruiushchikh kletok v énkhondral'nom ochage i osteogennye kletki-predshestvenniki.

It was shown by light and electron 3H-thymidine autoradiography that in the zones of endochondral osteogenesis in a tubular bone the little-differentiated perivascular cells are characterized by the most intensive proliferation among the DNA-synthesizing cells of the stroma. A comparison of the ultrastructural features and the distribution of labelled cells, their topographic interrelations at different times following the isotope injection suggests that during histogenesis in the endochondral centres the little-differentiated perivascular cells become spatially separated and are involved in the formation of the stromal cellular elements (in particular, fibroblasts, reticular cells, osteoblasts). In the developing and mature stroma, as well as in the endost, the little-differentiated forms of the population are preserved; the number of DNA-synthesizing cells among them decreases with age. It is suggested that the population includes stromal stem cells.