Non-resorbing osteoclasts induce migration and osteogenic differentiation of mesenchymal stem cells.

Osteoclast activity has traditionally been regarded as restricted to bone resorption but there is some evidence that also non-resorbing osteoclasts might influence osteoblast activity. The aim of the present study was to further investigate the hypothesis of an anabolic function of non-resorbing osteoclasts by investigating their capability to recruit mesenchymal stem cells (MSC) and to provoke their differentiation toward the osteogenic lineage. Bone-marrow-derived human MSC were exposed to conditioned media (CM) derived from non-resorbing osteoclast cultures, which were generated from human peripheral blood monocytes. Osteogenic marker genes (transcription factor Runx2, bone sialoprotein, alkaline phosphatase (AP), and osteopontin) were significantly increased. Osteogenic differentiation (OD) was also proved by von Kossa and AP staining occurred in the same range as in MSC cultures stimulated with osteogenic supplements. Chemotactic responses of MSC were measured with a modified Boyden chamber assay. CM from osteoclast cultures induced a strong migratory response in MSC, which was greatly reduced in the presence of an anti-human platelet-derived growth factor (PDGF) receptor beta antibody. Correspondingly, significantly increased PDGF-BB concentrations were measured in the CM using a PDGF-BB immunoassay. CM derived from mononuclear cell cultures did not provoke MSC differentiation and had a significantly lower migratory effect on MSC suggesting that the effects were specifically mediated by osteoclasts. In conclusion, it can be suggested that human non-resorbing osteoclasts induce migration and OD of MSC. While effects on MSC migration might be mainly due to PDGF-BB, the factors inducing OD remain to be elucidated.

[Effects of mechanical strain on human osteoblastic precursor cells in type I collagen matrices]. / Effekte mechanischer Reize auf humane osteoblastäre Zellen in einer dreidimensionalen Kollagen-Typ-I-Matrix.

BACKGROUND: The aim of the present study was to investigate the effect of mechanical strain on human osteoblastic precursor cells in a three-dimensional scaffold. METHODS: Osteoblastic precursor cells were seeded in a collagen type I gel and mechanically stretched by daily application of cyclic uniaxial strain. The expression of histone H4, core binding factor 1, alkaline phosphatase, osteopontin, osteocalcin, and collagen type I was investigated by analysing the mRNA. Cell and matrix orientation were investigated by scanning electron microscopy. RESULTS: Cyclic stretching increased cell proliferation. The expression of osteogenic markers was slightly increased by mechanical strain. The cells and matrix were strictly oriented in the stress direction. CONCLUSION: The application of mechanical load might have a beneficial effect on the quality and quantity of generated bone tissue and might be a important factor in tissue engineering of bone.

Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH.

Structural chromosome aberrations (SCAs) are sensitive indicators of a preceding exposure of the hematopoietic system to ionizing radiation. Cytogenetic investigations have therefore become routine tools for an assessment of absorbed radiation doses and their biological effects after occupational exposure or radiation accidents. Due to its speed and ease of use, fluorescence in situ hybridization (FISH) with whole chromosome painting (WCP) probes has become a method of choice to visualize SCAs. Until recently, this technique was limited to a rather small number of chromosomes, which could be tested simultaneously. As a result, only a fraction of the structural aberrations present in a sample could be detected and the overall dose effect had to be calculated by extrapolation. The recent introduction of two genome-wide screening techniques in tumor research, i.e., Spectral Karyotyping (SKY) and multicolor FISH (mFISH) now allows the detection of translocations involving any two non-homologous chromosomes. The present study was prompted by our desire to bring the power of mFISH to bear for the rapid identification of radiation-induced SCAs. We chose two model systems to investigate the utility of mFISH: lymphocytes that were exposed in vitro to 3 Gy photons and single hematopoietic progenitor cell colonies isolated from a Chernobyl victim 9 years after in vivo exposure to 5.4 Sv.In lymphocytes, we found up to 15 different chromosomes involved in rearrangements indicating complex radiation effects. Stable aberrations detected in hematopoietic cell colonies, on the other hand, showed involvement of up to three different chromosomes. These results demonstrated that mFISH is a rapid and powerful approach to detect and characterize radiation-induced SCAs in the hemopoietic system. The application of mFISH is expected to result in a more detailed and, thus, more informative picture of radiation effects. Eventually, this technique will allow researchers to rapidly delineate chromosomal breakpoints and facilitate the identification of the genes involved in radiation tumorigenesis.

Stable chromosomal aberrations in haemopoietic stem cells in the blood of radiation accident victims.

PURPOSE: The detection of long-term persistent chromosome aberrations in circulating haemopoietic stem cells after accidental radiation exposure. MATERIAL AND METHODS: Peripheral blood samples from highly exposed persons were collected 7-25 years after the radiation accidents in Moscow (1971), Kazan (1975) and Chernobyl (1996). Haemopoietic blood stem cells were analysed when investigating individual colonies derived from haemopoietic progenitor cells: burst-forming units-erythroid (BFU-E), granulocyte-macrophage-colony-forming cells (GM-CFC) and multipotent granulocyte-erythrocyte-macrophage- megakaryocyte-colony-forming cells (GEMM-CFC). Colony formation was obtained in methylcellulose cultures. Chromosome preparations in single colonies were performed using a microtechnique. RESULTS: Nine patients were investigated at 1 to 4 follow-up time points after radiation exposure. Three hundred and thirty-four single colonies were analyzed resulting in 1375 mitoses. It was found that colonies showed chromosome aberrations (ChA) up to 25 years after radiation exposure by classical cytogenetics and by fluorescence in situ hybridization (FISH). Stable aberrations were detected in 21% of colonies. They were clonal in 19% of colonies, i.e. the same abnormality was found in all cells derived from a single colony. In 2% of colonies ChA were stable but non-clonal; unstable ChA were not observed. CONCLUSIONS: The results indicate that blood-derived haemopoietic stem cells may serve as a biological indicator to detect radiation-induced ChA. Since they are considered to be in dynamic and functional exchange with stem cells in the medullary sites of blood cell formation such as bone marrow, the use of blood stem cells as a marker of radiation effects should be explored to assess the repair status of the stem cell pool as such.

Hemopoietic progenitor cells in the blood as indicators of the functional status of the bone marrow after total-body and partial-body irradiation: experiences from studies in dogs.

The granulocyte-macrophage colony-forming cells (GM-CFC) were studied in the blood of dogs to evaluate their relationship to the bone marrow GM-CFC under normal conditions and their involvement in hemopoietic regeneration after different types of exposure to ionizing radiation. The GM-CFC could be defined as regular blood elements showing characteristic levels of their concentration in individual dogs in the range from 20 to 300 cells per ml. In relative terms, the GM-CFC numbers present in the whole blood of normal dogs were found to be on the order of 0.1% of the GM-CFC numbers present in the bone marrow. A small fraction of the GM-CFC population in the bone marrow, i.e., about 1%, can be mobilized into the peripheral blood within three h by intravenous injection of dextran sulfate (DS). These cells are characterized by a small size and a low S-phase fraction similar to the GM-CFC that are normally present in the blood. Total-body irradiation with single doses of 0.8 Gy and more caused a characteristic pattern of sequential changes in the blood GM-CFC concentration that were related to the recovery of the bone marrow GM-CFC population. The blood GM-CFC concentration showed an extreme depression within the first 15 days, a transient increase from day 17 to day 35 and remained at subnormal values for several weeks and months. The regeneration of the GM-CFC population in the bone marrow that could be mobilized into the blood by DS was similarly delayed as the recovery of the blood GM-CFC values. In dogs which were kept under continuous radiation exposure (0.019 Gy/day) causing permanent damage to the hemopoietic system, the GM-CFC numbers in the blood remained permanently depressed. Partial-body irradiation of dogs with a myeloablative dose (11.7 Gy) given to the anterior part of their body was followed by sequential changes in the blood GM-CFC concentration specific for this type of exposure. The pattern of changes was determined by direct radiation effects, the compensatory responses in the protected bone marrow and the regeneration events in the irradiated bone marrow. On the other hand, it could be shown that the repopulation and the restoration of the hemopoietic tissue is initiated by the seeding of hemopoietic cells (including GM-CFC) from the protected marrow.

Acceleration of hemopoietic recovery in dogs after extended-field partial-body irradiation by treatment with colony-stimulating factors: rhG-CSF and rhGM-CSF.

PURPOSE: The influence of treatment with the two colony-stimulating factors, rhG-CSF and rhGM-CSF, on the hemopoietic recovery in aplastic bone marrow sites after extended-field irradiation was studied in a canine model. METHODS AND MATERIALS: The dogs received irradiation of the cranial part of their body with a single dose of 11.7 Gy, comprising approximately 72% of the total bone marrow mass. Anatomically this type of exposure corresponds to upper body irradiation (UBI) as employed under clinical conditions. Treatment with both the CSFs was employed for 7 days by daily injections of 30 microg/kg, starting 24 hr after irradiation. RESULTS: Treatment with rhGM-CSF did not completely prevent the initial decrease of the granulocyte counts, but caused an accelerated, though incomplete, recovery in the period from day 5 to day 15. In contrast, treatment with rhG-CSF caused two phases of granulocytosis and an early recovery to normal levels at day 11 after irradiation. Treatment with rhG-CSF, but not with rhGM-CSF, was associated with a strong supra-normal increase of progenitor cells in the blood within the first 8 days and an accelerated hemopoietic recovery in the irradiated sites particularly within the first 7 days after the exposure. CONCLUSIONS: These results indicate that under conditions of partial-body irradiation short term treatment with G-CSF is superior to GM-CSF in initiating the hemopoietic recovery on the basis of endogenous stem cell seeding.

Investigation of megakaryopoiesis in myelosuppressed bone marrow using immunogold-silver staining (IGSS).

To determine the frequencies and differential counts of megakaryocytes after cytoreductive treatment in nucleated low-density (1.060 g/ml) bone marrow cells (BMNC) of dogs an immunogold-silver staining (IGSS) technique with the lineage specific monoclonal antibody 2F9 was established. This antibody recognizes the glycoprotein IIb/IIIa complex expressed on the surface of canine megakaryocytes and platelets. The IGSS technique enables not only the detection of megakaryocytes occurring at a low frequency (0.1-0.2%), but also the discrimination between the different maturation stages of megakaryocytes due to cell size, nuclear morphology and cytoplasmic staining. By the use of this technique, small lymphoid megakaryocytic cells were identified. Comparable numbers of megakaryocyte colony-forming cells in 2F9-depleted and nondepleted BMNC suspensions (25.7 +/- 5.0 vs. 25.3 +/- 5.1 Meg-CFC/10(5) BMNC) indicate that these small 2F9 positive cells are nonclonogenic precursors of megakaryoblasts. To prove the applicability of IGSS, serial examinations of bone marrow samples from dogs treated with recombinant human interleukin-6 (IL-6) after exposure to 2.4 Gy total body irradiation (TBI) were performed. The results of the microscopic evaluation indicate that, in the recovery phase after TBI, IL-6 induced an earlier and stronger increase in megakaryocyte frequency in comparison to the control. Interestingly, all maturation stages of the megakaryocytic lineage took part in this IL-6 induced improvement of megakaryocyte recovery.

Assessment of DNA damage in canine peripheral blood and bone marrow after total body irradiation using the single-cell gel electrophoresis technique.

DNA damage in single peripheral blood (pb) and bone marrow (bm) cells was studied in dogs which were exposed to total body X-ray irradiation (TBI) with a lethal dose of 3.9 Gy. The changes in pb and bm cell numbers were measured within 9 days after TBI. Using the alkaline single-cell gel electrophoresis technique ('comet' assay), DNA strand breaks and alkali labile sites were assessed in single cells derived from the blood before TBI, 1 h and 4 h after TBI and on days 1, 3 and 9 after TBI. Bone marrow cells subjected to the assay were collected before and on days 1 and 9 after TBI. Cells expressing the strongest DNA damage were most frequent in the blood 1 h after TBI and in the bone marrow 1 day after exposure. Thereafter, a continuous reduction of DNA damage in individual cells was observed in the course of progressive leukopenia and granulocytopenia.

Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay.

Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose-effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D0 value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D0 values= 0.12-0.60 Gy. In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkali-labile sites was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stomal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as "comets" by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells.

Hematologic effects of recombinant human interleukin-6 in dogs exposed to a total-body radiation dose of 2.4 Gy.

The hematologic effects of recombinant human interleukin-6 (rhIL-6) were studied in dogs exposed to a total-body irradiation (TBI) of 2.4 Gy. IL-6 was administered over a period of 14 days at a daily dose of 18 micrograms/kg by single subcutaneous injection. Treatment was started 1 day after TBI. The data obtained for the different hematologic parameters of the irradiated IL-6-treated dogs were compared with the data obtained from dogs who received TBI of 2.4 Gy and were treated with the carrier (control). No clear influence of IL-6 treatment on the pattern of recovery of lymphocytes could be detected in comparison to the irradiated control animals. The thrombocyte counts in the period from day 1 to 16 after TBI were similar for both groups of dogs, showing a sharp decrease in counts between days 6 and 12 with a stabilization thereafter at approximately 30 x 10(3)/microL. In three of the four IL-6-treated dogs, however, thrombocyte counts increased at day 18 after the beginning of treatment. This increase occurred 7 days earlier than in the controls. In two of the three dogs showing an accelerated recovery of platelet counts, however, treatment with IL-6 caused a strong decrease in the erythrocyte counts associated with a prolonged depression in reticulocyte concentration. There was no influence on the recovery of blood granulocytes. In one of the animals responding with an accelerated thrombocyte recovery, IL-6 had no adverse effect on erythropoiesis. However, IL-6 forced the recovery of blood granulocytes in the period beyond day 10 after TBI. Another animal showed no influence of IL-6 on thrombocyte recovery but a strong depressive effect on erythrocyte and reticulocyte counts. The results show that for standardized conditions of radiation-induced bone marrow damage, the pattern of response to IL-6 in different hematopoietic lineages may show considerable variations between individuals, in contrast to what has been observed in irradiated animals treated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM- or G-CSF).

The effect of recombinant human stem cell factor and basic fibroblast growth factor on the in vitro radiosensitivity of CD34+ hematopoietic progenitors from human umbilical cord blood.

Human umbilical cord blood (CB) cells selected by immunomagnetic beads for expression of the CD34 antigen were irradiated with increasing doses of x-rays (72 cGy/min). Clonogenic survival of the hematopoietic progenitors, including mixed colony-forming cells (Mix-CFC), erythroid burst-forming units (BFU-E), and granulocyte-macrophage colony-forming cells (GM-CFC), was determined in methylcellulose cultures containing placenta conditioned medium (PCM) and erythropoietin (Epo). Exponential survival curves were fitted to the data of all the colonies, resulting in D0 = 95 cGy for Mix-CFC, 136 cGy for BFU-E, and 136 cGy for GM-CFC. Additionally, the radiosensitivity of CD34+ cells was studied employing cultures containing either recombinant human stem cell factor (rhSCF) or basic fibroblast growth factor (b-FGF) in combination with PCM and Epo. It was found that the colony-forming efficiency (CFE) of non-irradiated CD34+ cells of 5.5% (range 1.4 to 14.4%) did not increase after the addition of SCF or b-FGF to the culture. The radiation response characteristics showed, however, that in the presence of SCF, the D0 value and the extrapolation number n increased significantly. This suggests the stimulation of what operationally is termed "recovery from potentially lethal damage." In contrast, no response modifying effect could be seen for b-FGF.

Effects of total-body irradiation on bone marrow erythroid burst-forming units (BFU-E) and hemopoietic regeneration in dogs.

The acute and long-term effects of total-body X irradiation (TBI) on early erythroid progenitors, burst-forming units (BFU-E), in the bone marrow of beagles were studied for midline tissue doses of 1.6 and 2.4 Gy. After both radiation doses the initial reduction in the concentration of BFU-E was greater than that found for the granulocyte-macrophage progenitor cells (GM-CFC) and thus was in general agreement with the higher in vitro radiosensitivity of BFU-E compared to GM-CFC. After TBI with 1.6 Gy the GM-CFC and BFU-E returned to their normal levels within 2-4 weeks without showing long-term radiation effects. In contrast, after TBI with 2.4 Gy the concentrations of GM-CFC and BFU-E remained below the pretreatment levels up to 1 year after exposure. For a given midline tissue dose, the extent of the long-term effect of radiation on the BFU-E in a certain bone marrow site seems to be dependent on the local radiation dose in the respective bone marrow space. The minor radiation effects observed in the erythrocyte concentration in the peripheral blood, the hematocrit, and the hemoglobin concentration point to the enormous compensatory capacity of the more mature erythropoietic transit population to increase the proliferative capacity upon demand.

Haematological effects of rhGM-CSF in dogs exposed to total-body irradiation with a dose of 2.4 Gy.

It was the specific aim of this study to test the stimulatory effects of recombinant human GM-CSF (rhGM-CSF) on haemopoietic regeneration in dogs which had received total-body irradiation (TBI) with a dose of 2.4 Gy. In normal dogs rhGM-CSF given subcutaneously at 10 microgram/kg per day or 30 microgram/kg per day for 21 days caused strong but transient increases in the peripheral blood neutrophils. The monocyte counts also showed a transient rise during treatment in a dose-dependent fashion, whereas the lymphocyte counts increased only at the higher dose of rhGM-CSF and the platelet counts were transiently depressed during the course of the treatment. In the irradiated animals treatment with rhGM-CSF decreased the severity and shortened the duration of neutropenia but had no significant influence on monocyte or lymphocyte recovery. The granulocyte values showed a characteristic pattern of fluctuations with the first peak occurring at the same time (day 10 to day 13) when the abortive rise was observed in the untreated dogs. In contrast the GM-CFC in the peripheral blood remained depressed during the whole treatment course, similar to the untreated irradiated controls. These results indicate that treatment with GM-CSF can be an effective biological monotherapy for radiation-induced bone marrow failure, but that for higher radiation doses the number of GM-CSF responsive target cells will become a critical determinant of therapeutic efficacy.

In vitro studies on the radiosensitivity of multipotent hemopoietic progenitors in canine bone marrow.

The in vitro radiation response to 280-kV x-rays (does rate 72 cGy/min) of multipotent hemopoietic progenitor cells, mixed colony-forming units (CFU-mix), from canine bone marrow was assayed and compared to the radiation response characteristics of early erythroid progenitors, erythroid burst-forming units (BFU-E). To improve the colony-forming efficiency, the effect of various bone marrow cell separation techniques on colony formation of both progenitors was examined. The separation of bone marrow aspirates by discontinuous buoyant gradient centrifugation using the lymphocyte separation medium Lymphoprep with a density of 1.070 g/ml allowed the establishment of reproducible survival curves. The survival curves for both progenitors were strictly exponential, and CFU-mix were found to be more radiosensitive (D0 = 12 +/- 2 cGy) than BFU-E (D0 = 16 +/- 2 cGy).

Thymic nurse cells: differentiation of thymocytes within complexes.

Thymic nurse cell complexes (TNC-c) were isolated from thymuses of BDF1 mice at pre-determined intervals during the 12-week latency period that precedes the development of leukemias. T-cell leukemias were induced by a single i.v. injection of 50 mg/kg of methylnitrosourea (MNU). In order to clarify processes taking place in TNC-c, the complexes of mice after MNU injection were compared with TNC-c of age-matched control mice, with respect to their number per thymus, the distribution of TNC-c according to their size (the number of intra-TNC thymocytes reflects the type of TNC-c), the number of intra-TNC thymocytes that undergo DNA synthesis, and the phenotype of thymocytes inside TNC-c. During the latency period of leukemogenesis, the effects of MNU were shown to involve, in addition to changes in number of TNC-c, a decrease in the number of thymocytes incorporating labeled thymidine, viz., the number of dividing cells, thus affecting the size distribution of TNC-c types. Intra-TNC thymocytes of control mice were heterogeneous in their phenotype and represented cells at varying stages of their maturation cycle. MNU administration was followed by selective differentiation of thymocytes within TNC-c to Lyt 1-thymocytes in some and to Lyt 2-thymocytes in others. Lyt 1 and Lyt 2 being specific antigens expressed by thymocytes.

In vitro studies of the sensitivity of canine bone-marrow erythroid burst-forming units (BFU-E) and fibroblast colony-forming units (CFU-F) to X-irradiation.

The radiosensitivity of the early erythroid progenitor cells (BFU-E) and the progenitor cells of the stroma (CFU-F) in canine bone marrow was studied under steady-state conditions by in vitro irradiation with 280 kV X-rays. The dose-effect relationship for colony formation was determined for BFU-E obtained from the iliac crest marrow, and for CFU-F in bone marrow collected from the iliac crest and the humerus of adult beagles. The BFU-E were adequately stimulated with serum from lethally irradiated dogs to obtain a source of BPA (burst-promoting activity). The BFU-E proved to be extremely radiosensitive, and the survival curve was exponential (D0 = 15.3 +/- 1.8 cGy). We showed that buffy-coat leukocytes separated from bone marrow leukocytes obtained by aspiration were an optimum source of CFU-F. A curve was fitted to the data obtained for CFU-F obtained from the iliac crest or the humerus, resulting in D0 = 241 +/- 38 cGy and an extrapolation number n = 1.38 +/- 0.62 or D0 = 261 +/- 40 cGy and n = 1.04 +/- 0.42, respectively. According to these findings, and other published data, we conclude that the canine bone marrow BFU-E are presently the most radiosensitive hemopoietic cells detected among all hemopoietic cells of different mammals.

Growth of erythroid burst-forming units (BFU-E) in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs.

Erythroid burst-forming units (BFU-E) from canine bone marrow and peripheral blood could be grown in methylcellulose in the presence of an appropriate batch of fetal calf serum (FCS), transferrin, and erythropoietin (Epo). However, improved colony formation (size and number of bursts) was obtained when serum from total body irradiated dogs was present in the culture. This serum, obtained from dogs at day 9 after total body irradiation with a dose of 3.9 Gy, reduced markedly the Epo requirement of BFU-E. Furthermore, it allowed the omission of FCS from the culture medium if cholesterol and bovine serum albumin (BSA) were used as FCS substitutes. BFU-E concentrations were found to be rather different in the peripheral blood and in bone marrow samples from different sites (i.e., iliac crest, sternum, and humerus) of normal beagles. The studies further show that canine bone marrow BFU-E can be cryopreserved in liquid nitrogen.

Murine thymic multicellular complexes during latency period after methylnitrosourea (MNU) injection.

The number of TNC complexes and of stroma cell-thymocyte rosettes was examined during the preleukemic period in mice given MNU to induce leukemia. In parallel, numbers of the complexes were studied following administration of hydrocortisone one day before administering MNU, i.e. after the procedure which clearly inhibited manifestation of induced leukemias. Administration of MNU with or without hydrocortisone is followed by disappearance of TNC complexes and stroma cell-thymocyte rosettes followed by their regeneration between 2 and 6 weeks after MNU. Relative rates of regeneration were different in MNU treated versus MNU + hydrocortisone treated animals.

Trisomy 15 as a regular finding in chemically induced murine T-cell leukemogenesis.

Trisomy 15 is described as a common finding in all T-cell leukemias induced by a single dose of methylnitrosourea (MNU) in BDF1 mice and in the leukemias induced by 7 doses of benzo(a)pyrene. Additional trisomies were found in about half of the leukemias. The organ distribution suggests that the leukemic cells with trisomy 15 originate in the thymus. Trisomy 15 was detected in the thymus as early as 6 weeks after the application of MNU, i.e. during the latency period.