Kinetic-microarchitectural correlations in the bone marrow of the mouse.

Transverse histologic sections of bone marrow obtained from mice that were sacrificed by perfusion fixation at intervals following tritiated thymidine injection were studied by means of radioautography. A kinetic gradient was demonstrated across the marrow section, with the highest proliferative rate in the subendosteal region. Megakaryocytes were shown to originate from the rapidly proliferating subendosteal cells. The immediate proliferating precursors of mature granulocytes were slowly proliferating cells found predominantly in the central region of the marrow. It was concluded that in the steady state there must be a migration of cells from the subendosteal region to the central region with concomitant growth retardation of the migrating cells.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

Elimination of T lymphocytes from the bone marrow in HLA-incompatible bone marrow transplantation

The method presented is very well suited to eliminate T-lymphocytes from great amounts of bone-marrow. The stem cells required to reconstitute the bone-marrow are enriched in this way. It can be completely performed in a closed system. Any contamination with germs is excluded. It can be reproduced well and learnt quickly. It takes 10 hours for two trained co-workers to process 1,500 ml of bone-marrow. The vitality of cells is very good (100%). Its suitability for transplantation has still to be checked.     

Stromal cells of the bone marrow in growing bone

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.     

Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.     

Registry of unrelated bone marrow donors.

Reports of successful transplantation of bone marrow obtained from unrelated donors who were histocompatibility leukocyte antigen (HLA) identical prompted the Canadian Red Cross Blood Transfusion Service in Ottawa to assess the possibility of developing a bone marrow donor registry in Canada. We sent a pamphlet that explained the program to 1568 people who had undergone apheresis and asked them to reply, stating their interest. At the same time the pamphlets and a poster were placed in the blood donor clinic. We received 1232 replies (78.6%) from the apheresis donors, 838 (68.0%) of which indicated a willingness to attend information sessions. Of the 7158 people who gave blood during the 3-month study period, 225 (3.1%) were interested. At the time this paper was written 47 information sessions had been held, and 721 people had attended, 624 (86.5%) of whom had signed a consent form. This indicates a clear interest in a bone marrow donation program. We believe that the ethical issues are overcome by requesting the donation before identification of any patient. From our experience a national registry of unrelated donors seems feasible, and steps are being taken to implement such a program.     

Iron overload of the bone marrow by trimethylhexanoyl-ferrocene in rats.

Iron-deficient female Wistar rats were fed a diet which contained 0.5% 3,5,5-trimethylhexanoyl (TMH)-ferrocene over a 57-week period. The state of iron deficiency was characterized by means of the absence of stainable iron in the bone marrow. After the first days on the iron-enriched diet, ferritin-containing siderosomes were found, in numerous erythroblasts up to orthochromatic normoblasts and in reticulocytes, i.e. the dispensed iron was used for haemoglobin synthesis. After 1 week the first macrophages showed a positive Perls' Prussian blue reaction. In the cytoplasm they stored the iron in the form of free ferritin molecules and lysosomally as aggregated ferritin and/or haemosiderin. The iron loading of the macrophages increased in both of the storage qualities proportionally with duration of the feeding period and reached a maximum after 38 weeks. Final stages showed extremely iron-loaded macrophages with high concentrations of free ferritin molecules and large siderosomes, partially flowing together to still greater units. Iron deposits within endothelial cells of bone marrow sinusoids can be observed for the first time after 4 weeks. In these cells the iron is stored as ferritin in siderosomes of relatively small and uniform size; free ferritin molecules in the cytosol were of only slight concentration. The TMH-ferrocene model of iron overload shows in the bone marrow: (1) an unimpeded utilization of the iron component for erythropoiesis, (2) development of excessive iron overload of the bone marrow in macrophages and endothelial cells of sinusoids and (3) a pattern of distribution of iron as seen in secondary haemochromatosis.