Rapid inactivation and apoptosis of osteoclasts in the maternal skeleton during the bone remodeling reversal at the end of lactation

There is a rapid reversal in maternal skeletal metabolism and bone remodeling from accelerated bone resorption during lactation to skeletal rebuilding after lactation. The purpose was to determine the changes that occur in maternal osteoclasts during the transition from lactation to postlactation. Skeletal samples were taken from female rats on days 10 and 19 of lactation and 1 and 7 days after lactation. The pups were weaned on day 20. There was a rapid change in the osteoclast population after weaning, resulting in less resorption surface. Osteoclasts detached from bone surfaces, lost their ruffled borders, and became fragmented with immunocytochemical evidence of apoptosis within 24 hr after lactation. Concomitant with the rapid regression in the osteoclast population was an over fivefold increase in maternal calcitonin (CT) levels at 24 hr after weaning. Serum calcium and estrogen (E2) increased, but prolactin (PRL) and PTH decreased after weaning. The hormone changes, particularly that of CT, are consistent with the rapid regression of the osteoclast population at the end of lactation. These changes are similar to a reversal phase of a bone remodeling cycle where bone formation commences when resorption ceases on bone surfaces and suggests that the fate of osteoclasts during bone remodeling is programmed cell death. These results also suggest that bone remodeling is well synchronized prior to, during, and after lactation to accommodate the mineral requirements of the offspring as well as the mother. Anat Rec, 290:65–73, 2007. © 2006 Wiley‐Liss, Inc.     

Topography and behavior of Sertoli cells in sparse culture during the transitional remodeling phase

We report observations on the behavior of Sertoli cells in sparse culture during the period from the time of plating to the time of initial confluence (the transitional remodeling phase). Changes in shape, structure, and polarity of cells, as well as changes in migration patterns and cell-cell association patterns, have been followed during the transitional remodeling phase with the aid of topographical markers. These markers are based upon differences between ultrastructural features of the basolateral and apicolateral surfaces. The basolateral surface is characterized by plasmalemmal blebs, whereas the apicolateral surface is characterized by filopodial extensions. Structural differences observed in situ remain evident in Sertoli cells isolated by sequential enzymatic treatments that are described. Another marker is provided by laminin-binding sites, which are detected exclusively on the blebbed, basolateral surfaces of freshly prepared Sertoli cell aggregates. The orientation described is sustained during the initial radial migration of Sertoli cells explanted on uncoated glass coverslips. Under these conditions, blebs are detected only on the dorsal surfaces, and filopodial extensions are evident only on the ventral surfaces. In contrast, Sertoli cells sparsely plated on a reconstituted basement membrane (air-dried Matrigel) migrate rapidly, display an extraordinary capacity to form elaborate cytoplasmic extensions for cell-cell and cell-substratum contacts, and readily retract blebs and filopodial extensions. These cells do not form mosaic borders, whereas cells plated on uncoated glass do form a monolayer with mosaic-like borders. Cells sparsely seeded on gelated Matrigel migrate preferentially at gaps between adjacent cell explants, and develop a compact cell-cell association pattern. These cells display few, if any, cytoplasmic extensions. We compare the behavior of Sertoli cells sparsely plated on Matrigel with the behavior of Sertoli cells in situ during different stages of development.     

Arterial remodeling following mechanical injury. The role and nature of smooth muscle cells

The main feature of an atherosclerotic plaque is the formation of a new tissue in the arteries. In this respect atherosclerosis is similar to other conditions where non-neoplastic tissue formation occurs like in embryogenesis, in healing or in repair processes. A progressive intimal thickening occurs in the early phase of human atherosclerotic lesions and also in certain experimental models. Long-standing aortic intimal thickening could be induced by mechanical injury to the inner surface of the aorta with a microsurgical instrument, which causes controlled endothelial denudation. The injury is followed by an arterial remodeling. The latter process is caused by the development of an intimal plaque which consists of two main components: the smooth muscle cell (SMC) and the intercellular matrix. The matrix components are mostly synthetized by the SMC. Two distinct SMC populations could be distinguished by morphological means in the intimal proliferation: the synthetizing type and the proliferating type. Their role will be discussed and their morphological appearance will be compared with SMC present in other lesions.     

Effects of bone marrow-derived mononuclear cells on airway and lung parenchyma remodeling in a murine model of chronic allergic inflammation

We hypothesized that bone marrow-derived mononuclear cells (BMDMC) would attenuate the remodeling process in a chronic allergic inflammation model. C57BL/6 mice were assigned to two groups. In OVA, mice were sensitized and repeatedly challenged with ovalbumin. Control mice (C) received saline under the same protocol. C and OVA were further randomized to receive BMDMC (2 × 10⁶) or saline intravenously 24 h before the first challenge. BMDMC therapy reduced eosinophil infiltration, smooth muscle-specific actin expression, subepithelial fibrosis, and myocyte hypertrophy and hyperplasia, thus causing a decrease in airway hyperresponsiveness and lung mechanical parameters. BMDMC from green fluorescent protein (GFP)-transgenic mice transplanted into GFP-negative mice yielded lower engraftment in OVA. BMDMC increased insulin-like growth factor expression, but reduced interleukin-5, transforming growth factor-β, platelet-derived growth factor, and vascular endothelial growth factor mRNA expression. In conclusion, in the present chronic allergic inflammation model, BMDMC therapy was an effective pre-treatment protocol that potentiated airway epithelial cell repair and prevented inflammatory and remodeling processes.     

Distribution of mononuclear cells during pregnancy.

Leucocyte and lymphocyte counts, the distribution of erythrocyte rosette-forming cells (E-RFC), erythrocyte-antibody-complement rosette-forming cells (EAC-RFC), cells bearing surface membrane immunoglobulin (SMIg), latex-ingesting cells and the PHA stimulation index of lymphocytes were evaluated in both pregnant and non-pregnant women. In the gravid female leucocytosis due to neutrophilia is seen, and the PHA-induced transformation of maternal lymphocytes in the absence of autologous plasma remains unaltered. There was a rise in the percentage of EAC-RFC and cells bearing SMIg during a pregnancy, but after monocyte adsorption on plastic dishes of the mononuclear cells, obtained after Ficoll-Hypapue centrifugation, this increase is abolished. There is a significant increase of the number of latex-ingesting cells during pregnancy. These findings indicate a significant increase of the monocytes during pregnancy.     

Functional activity of blood mononuclear cells during regeneration of bone tissue

It is established that, during elongation of a segment of the extremity, normal regeneration of the bone tissue is attended by a slight decrease in monocyte adherence during the postoperative period and by an increase of the functional activity of lymphocytes during subsequent activation of osteogenesis. Suppression of the lymphoid apparatus is observed in the case of disturbed osteogenesis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 1117, N ^o 1, pp. 104–105, January, 1994     

Cytotoxic effector capacity of bone marrow mononuclear cells.

Purified mononuclear cells from guinea pig bone marrow possess highly efficient cytotoxic effector cell capabilities in the phytohemagglutinin-induced cellular cytotoxity and antibody-dependent cellular cytotoxicity and antibody-dependent cellular cytotoxicity assays against chicken red blood cell targets. This cytotoxic activity is not removed by depletion of adherent cells by passage through rayon wool columns. Thus bone marrow lymphocytes themselves, depleted of adherent monocytes, possess this killer cell capacity. These effector cells may either arise de novo within the bone marrow parenchyma or arrive there as part of the recirculating lymphocyte pool. These studies lend further understanding of the development of functional capabilities of bone marrow lymphoid cells.     

Theta-bearing cells in the bone marrow of thymus-deprived mice. Numbers and nature.

Theta-bearing cells in lymphomyeloid tissues of thymus-deprived and normal mice have been studied by the use of anti-theta antiserum and cytotoxicity tests in addition to functional tests. In contrast to the findings in peripheral lymphoid tissues, increased percentages and numbers of theta-bearing cells were found in the bone marrow of neonatally and nude mice as compared with normal and sham-thymectomized mice. In adult thymectomized mice, percentages comparable to those in sham-operated littermates were found. The findings were not due to irrelevant antibodies in the anti-theta antiserum, and neonatally thymectomized mice grafted with a thymic lobe showed percentages of theta-positive cells in the bone marrow comparable to those of sham-operated animals. Adrenalectomy did not lead to diminished percentages of theta-positive cells in the bone marrow of neonatally thymectomized mice, and the serum levels of hydrocortisone and corticosterone were within normal ranges in thymus-deprived mice. The mitogen responses and graft-versus-host activity of bone marrow cells from neonatally thymectomized mice suggest that most theta-positive cells in the bone marrow of these mice are functionally immature cells.     

The thiol-proteindisulfide oxidoreductase in human mononuclear cells of blood and bone marrow

The in vivo function of the thiol-proteindisulfide oxidoreductase (TPO, EC 1.8.4.2; proteindisulfide isomerase, EC 5.3.4.1) in biosynthesis of immunoglobulin was investigated by studying the enzyme content in human lymphoid and other cells by an immunocytochemical method. In contrast to peripheral blood, B lymphocytes which showed no or no demonstrable TPO, normal as well as malignant bone marrow plasma cells (all Ig classes) were found to contain abundant amounts of this enzyme. TPO containing plasma cells were identified by double-staining techniques. This finding suggests that TPO is involved in the terminal step of B cell differentiation and immunoglobulin biosynthesis. Besides plasma cells, approximately 10% of mononuclear marrow cells as yet unidentified medium-sized and large cells, exhibited also strong anti-TPO reactivity. Furthermore, using surface-cytoplasmic double staining methods, monocytes from human peripheral blood could be identified to represent the only cytoplasmic TPO-containing normal mononuclear blood cells.     

Expression of interleukin-8 by lipopolysaccharide-stimulated bone marrow-derived mononuclear cells.

Interleukin-8 (IL-8) is a potent neutrophil chemoattractant, produced by a variety of immune and nonimmune cells in response to exogenous and host-derived inflammatory stimuli. We demonstrate here that a suspension of normal bone marrow mononuclear cells, consisting principally of myeloid precursors, produces IL-8 in response to stimulation with lipopolysaccharide (LPS). IL-8-specific mRNA is rapidly induced, being detected first 30 min after stimulation. IL-8 is detected by enzyme-linked immunosorbent assay within 2 h of stimulation, with steady a increase in its level through 72 h. Further studies demonstrated that LPS could serve as a primary stimulus for the expression of IL-8, since LPS challenge in the presence of cycloheximide resulted in superinduction of bone marrow mononuclear cell-derived IL-8 mRNA. These investigations suggest that the stimulatory effect of LPS is independent of other cytokines such as IL-1 beta. When compared with LPS, IL-1 beta proved to be a weak signal for the expression of IL-8 by bone marrow mononuclear cells. In a dose-response study, the maximum stimulatory concentration of IL-1 beta (300 pg/ml) resulted in the production of 500 pg of IL-8 per 10(6) cells, whereas 1 microgram of LPS resulted in the production of 5.5 ng/10(6) cells. Although IL-1 beta was not a particularly potent stimulus for IL-8 production by bone marrow mononuclear cells, peripheral blood mononuclear cells were highly susceptible to IL-1 beta challenge. In addition, the potential dependence of LPS-induced marrow-derived IL-8 production on the intermediate synthesis of IL-1 beta was further investigated. Results of studies assessing kinetics, addition of cycloheximide, and blocking with IL-1 beta neutralizing antibody were all consistent with the ability of LPS to directly induce bone marrow-derived IL-8 independently of IL-1 beta. These investigations demonstrate that bone marrow may be a significant source of IL-8 and may play a significant role in acute infectious, inflammatory responses.     

Independent regulation of ABCB1 and ABCC activities in thymocytes and bone marrow mononuclear cells during aging

Aging modifies a number of functional and phenotypic parameters of cells from the immune system. In this study, the activities of two members of the superfamily of ATP-binding cassette (ABC) transport proteins, ABCB1 and ABCC (measured by rhodamine 123 efflux and Fluo-3 efflux respectively), were compared in murine bone marrow cells and thymocytes of young (3-4 weeks old), adult (2-3 months old) and old (18 months old) mice. ABCB1 activity was shown to be age regulated in murine bone marrow mononuclear cells and thymocytes. In the bone marrow, the increased amount of cells with ABCB1 activity observed in old mice was restricted to the c-kit(-)Sca-1(+) and c-kit(+)Sca-1(+) subpopulations. Only a small percentage of c-kit(+) cells in the thymus had ABCB1 activity, and this subpopulation increased with age. In the thymus, old age augmented this activity in the CD4(-) CD8(-) double-negative cells and in the CD4(+) and CD8(+) single-positive populations. The activity of another ABC transporter, the ABCC-related activity, was also modified by age in the bone marrow. However, the age-related increase was observed in the subpopulations were ABCB1 was not modified, namely the non-progenitor population (c-kit(-)Sca-1(-)cells) and c-kit(+)Sca-1(-) cells. Nearly, all thymocytes expressed the ABCC1 molecule in an active form and aging did not affect this pattern. This study demonstrates an independent upregulation of ABCB1 and ABCC activities during the aging process. The increases were observed in different subsets of cells but followed a developmentally regulated pattern. The functions played by these transporters and alterations in aging are discussed.     

Neovascularization and bone regeneration by implantation of autologous bone marrow mononuclear cells

We examined whether transplantation of autologous bone marrow mononuclear cells (BM-MNCs) can augment neovascularization and bone regeneration of bone marrow in femoral bone defects of rabbits. Gelatin microspheres containing basic fibroblast growth factor (bFGF) were prepared for the controlled release of bFGF. To evaluate the in vivo effect of implanted BM-MNCs, we created bone defects in the rabbit medial femoral condyle, and implanted into them 5 x 10(6) fluorescent-labeled autologous BM-MNCs together with gelatin microspheres containing 10 microg bFGF on an atelocollagen gel scaffold. The four experimental groups, which were Atelocollagen gel (Col), Col + 5 x 10(6) BM-MNCs, Col + 10 microg bFGF, and Col + 5 x 10(6) BM-MNCs + 10 microg bFGF, were implanted into the sites of the prepared defects using Atelocollagen gel as a scaffold. The autologous BM-MNCs expressed CD31, an endothelial lineage cell marker, and induced efficient neovascularization at the implanted site 2 weeks after implantation. Capillary density in Col + BM-MNCs + bFGF was significantly large compared with other groups. This combination also enhanced regeneration of the bone defect after 8 weeks to a significantly greater extent than either BM-MNCs or bFGF on their own. In summary, these findings demonstrate that a combination of BM-MNCs and bFGF gelatin hydrogel enhance the neovascularization and the osteoinductive ability, resulting in bone regeneration.     

Human rib bone marrow mononuclear cells spontaneously synthesize and secrete IgE in vitro.

We have examined spontaneous secretion of IgE by human rib bone marrow mononuclear cells (MNC). Bone marrow MNC from nine out of 12 rib specimens synthesized and secreted substantial amounts of IgE during 14 days of in vitro culture. The 14-day supernatants from these bone marrow MNC contained a mean of 2589 pg/ml of IgE (n = 12) with a maximum production of 15,408 pg/ml of IgE compared with small amounts of IgE (80-200 pg/ml) produced by similarly cultured normal and inflammatory bowel disease intestinal lamina propria MNC. Using two rib specimens, time-course studies revealed spontaneous secretion of IgE to be minimal during the first 2 days of culture (152 pg/ml), followed by a steady increase between days 4 (517 pg/ml) and 14 (3588 pg/ml). The addition of pokeweed mitogen resulted in 72% suppression of spontaneous IgE production by bone marrow MNC. The bone marrow MNC isolated from the ribs consisted of 22% Leu12+ (B) cells of which 3.2% were surface IgE positive. Staining for cytoplasmic immunoglobulin revealed 1% of the bone marrow MNC to be cytoplasmic IgE+. The presence of IgE-bearing and IgE-secreting MNC in human bone marrow is consistent with the observation that allergen-specific IgE-mediated hypersensitivity is adoptively transferred by human bone marrow transplantation and demonstrates the usefulness of human bone marrow MNC for examination of IgE secretory and regulatory events.     

Influence of engineered titania nanotubular surfaces on bone cells

A goal of current orthopedic biomaterials research is to design implants that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, these implants should result in the formation of a characteristic interfacial layer with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the bone-material interface is needed, as well as the development of new materials and approaches that promote osseointegration. Using anodization, titania interfaces can be fabricated with controlled nanoarchitecture. This study demonstrates the ability of these surfaces to promote osteoblast differentiation and matrix production, and enhance short- and long-term osseointegration in vitro. Titania nanotubular surfaces were fabricated using an anodization technique. Marrow stromal cells (MSCs) were isolated from male Lewis rats and seeded on these surfaces along with control surfaces. The interaction of cells with these surfaces was investigated in terms of their ability to adhere, proliferate and differentiate on them. The experiments were repeated three times with cells from different cultures. All the results were analyzed using analysis of variance (ANOVA). Statistical significance was considered at p<0.05. Furthermore, in vivo biocompatibility was assessed by implanting surfaces subcutaneously in male Lewis rat and performing histological analysis after 4 weeks. Our results indicate that the nanotubular titania surfaces provide a favorable template for the growth and maintenance of bone cells. The cells cultured on nanotubular surfaces showed higher adhesion, proliferation, ALP activity and bone matrix deposition compared to those grown on flat titanium surfaces. In vivo biocompatibility results suggest that nanotubular titania does not cause chronic inflammation or fibrosis. The fabrication routes of titania nano-architectures are flexible and cost-effective, enabling realization of desired platform topologies on existing non-planar orthopedic implants.     

Comparative effects of Mycobacterium avium glycopeptidolipid and lipopeptide fragment on the function and ultrastructure of mononuclear cells.

Among the various lipids associated with the cell envelope of the Mycobacterium avium complex, the species-specific glycopeptidolipids (GPL) are responsible for distinguishing one serovar from another. In a continuing effort to study the immunomodulatory capabilities of these mycobacterial lipids, we have examined and compared the effects of the GPL and its lipopeptide fragment (beta-lipid) on mononuclear cell function. It was observed that the lymphoproliferative response of murine splenic mononuclear cells to mitogen stimulation was reduced by both the GPL and its lipopeptide fragment. Although the responsiveness appeared to be down-regulated to a greater degree by the beta-lipid, treatment with either GPL or beta-lipid resulted in the release of soluble factors from peritoneal macrophages that caused suppression of the lymphoproliferative responsiveness of splenic mononuclear cells. Flow cytometric analysis of peritoneal macrophages revealed that treatment with the beta-lipid fragment caused a marked decrease in expression of the C3bi complement receptor, Mac-1, on macrophages, whereas treatment with GPL resulted in a marked increase in the expression of Mac-2 receptor on macrophages. Treatment of peritoneal macrophages with either GPL or beta-lipid resulted in the release of tumour necrosis factor (TNF), as determined by an L929 biological cytotoxicity assay. Perturbation of macrophage membrane ultrastructure by both GPL and beta-lipid was confirmed by electron microscopy, and may be a possible explanation for the resulting alterations in mononuclear cell function observed in this study.     

The promotion of the vascularization of decalcified bone matrix in vivo by rabbit bone marrow mononuclear cell-derived endothelial cells

The neovascularization of bone grafting represents an important challenge in bone regeneration. Prevascularization of tissue-engineered bone using endothelial cells (ECs) in vitro sheds light on accelerating the vascularization of bone replacements. In the present study, decalcified bone matrix (DBM) was prevascularized by seeding fibrin gels with ECs that are derived from rabbit bone marrow mononuclear cells (BMMNCs). The compound was then transplanted autologously into bone defects of rabbits to observe the vascularization in vivo. At 2, 4 and 8 weeks after grafting, the microvessel density of new bone tissues was significantly higher in the experimental group than in the control group (P < 0.05), which suggests that prevascularization of BMMNC-derived cells may be suitable for improving vascularization in tissue-engineered bone.     

Immunophenotype of multinucleated and mononuclear cells in giant cell lesions of bone and soft tissue.

AIMS: To compare the antigenic phenotype of giant cells in giant cell lesions of bone and soft tissue with that of osteoclasts and macrophage polykaryons. METHODS: Formalin fixed, paraffin wax embedded sections of 106 giant cell lesions, 19 granulomatous, and 14 osteoclast containing lesions were immuno-histochemically stained for leucocyte common antigen (LCA), CD68, and HLA-DR. RESULTS: Osteoclasts and giant cells of giant cell tumour of bone and giant cell reparative granuloma could be distinguished by their generalised absence of HLA-DR reaction from macrophage polykaryons and giant cells in other giant cell lesions of bone and soft tissue. Staining for LCA, CD68, and HLA-DR was useful in distinguishing reactive histiocytic giant cells and osteoclasts from tumour giant cells. CONCLUSIONS: A panel of macrophage associated antigens should be diagnostically useful in differentiating the histological nature of giant cells in various giant cell lesions of bone and soft tissue.