Osteoblastic responses to TGF-beta during bone remodeling

Bone remodeling depends on the spatial and temporal coupling of bone formation by osteoblasts and bone resorption by osteoclasts; however, the molecular basis of these inductive interactions is unknown. We have previously shown that osteoblastic overexpression of TGF-beta2 in transgenic mice deregulates bone remodeling and leads to an age-dependent loss of bone mass that resembles high-turnover osteoporosis in humans. This phenotype implicates TGF-beta2 as a physiological regulator of bone remodeling and raises the question of how this single secreted factor regulates the functions of osteoblasts and osteoclasts and coordinates their opposing activities in vivo. To gain insight into the physiological role of TGF-beta in bone remodeling, we have now characterized the responses of osteoblasts to TGF-beta in these transgenic mice. We took advantage of the ability of alendronate to specifically inhibit bone resorption, the lack of osteoclast activity in c-fos-/- mice, and a new transgenic mouse line that expresses a dominant-negative form of the type II TGF-beta receptor in osteoblasts. Our results show that TGF-beta directly increases the steady-state rate of osteoblastic differentiation from osteoprogenitor cell to terminally differentiated osteocyte and thereby increases the final density of osteocytes embedded within bone matrix. Mice overexpressing TGF-beta2 also have increased rates of bone matrix formation; however, this activity does not result from a direct effect of TGF-beta on osteoblasts, but is more likely a homeostatic response to the increase in bone resorption caused by TGF-beta. Lastly, we find that osteoclastic activity contributes to the TGF-beta-induced increase in osteoblast differentiation at sites of bone resorption. These results suggest that TGF-beta is a physiological regulator of osteoblast differentiation and acts as a central component of the coupling of bone formation to resorption during bone remodeling.     

Stress increases the population of splenic macrophages, permissive for Langat virus, in mice

Cytogenetic study conducted after a single intraperitoneal injection of cyclophosphane in doses of 10, 20, and 40 mg/kg showed in 1.5-2-month-old male mice that the level of bone marrow cells damaged by the mutagen was significantly higher in NZW animals than in C57Bl/6 animals. The ability to produce active oxygen forms in response to addition of opsonized zymosan and phorbol myristate acetate was also higher in bone marrow suspensions of NZW mice. The higher sensitivity of NZW animals to pro-oxidant and clastogenic effects may be related to the genetically determined decrease of antioxidant protection in NZW animals.     

Effects of La~（3＋） on osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells

In order to elucidate the action of La3+ on bone metabolism,effects of La3+ on the osteogenic and adipogenic differentiation of pri-mary mouse bone marrow stromal cells(BMSCs) were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) test,alkaline phosphatase(ALP) activity measurement,mineralized function,oil red O stain and measurement.The results showed that La3+ pro-moted the proliferation of BMSCs except at 1×10-10 and 1×10-6 mol/L.The effect of La3+ on the osteogenic differentiation depended on con-centrations at the 7th day,but the osteogenic differentiation was inhibited at any concentration at the 14th day.La3+ promoted the formation of mineralized matrix nodules except at 1×10-8 and 1×10-5 mol/L.La3+ inhibited adipogenic differentiation except at 1×10-10 and 1×10-7 mol/L at the 10th day,and inhibited adipogenic differentiation except at 1×10-9 mol/L at the 16th day.These findings suggested that La3+ might have protective effect on bone at appropriate dose and time.This would be valuable for better understanding the mechanism of the effect of La3+ on bone metabolism.     

Assessment of basal sound identification skills and communication abilities in profoundly deaf children fitted with hearing aids or a cochlear implant

Recent in vitro findings suggest that bisphosphonates, potent inhibitors of osteoclastic bone resorption, may also have a direct action on osteoblasts. The purpose of this study was to search for potential effects of etidronate and alendronate on the formation of early and late osteoblastic cell precursors by measuring the number of colony-forming units for fibroblasts (CFU-F) and colony-forming units for osteoblasts (CFU-OB) in murine and human bone marrow cultures. In murine marrow cultures, etidronate (10(-5) to 10(-9) mol/L) significantly stimulated the formation of CFU-F with a maximal effect at 10(-5) mol/L (mean increase over control values+/-SD: 106+/-17%;p < 0.001), whereas alendronate had a biphasic effect, being stimulatory at concentrations below 10(-7) mol/L (78+/-5%; p < 0.001), and inhibitory at higher doses. The formation of CFU-OB was also inhibited by both bisphosphonates at the highest concentrations (10(-5) mol/L and 10(-6) mol/L), but it was significantly stimulated at lower concentrations (from 10(-7) to 10(-9) mol/L for etidronate and 10(-7) to 10(-10) mol/I, for alendronate; p < 0.001). In human bone marrow cultures, alendronate (10(-8) to 10-(12) mol/L) increased CFU-F formation with a maximal effect at 10(-10) mol/L (161+/-12 %; p < 0.01). CFU-OB formation, observed only in the presence of dexamethasone (10(-8) mol/L), was markedly stimulated by alendronate at the above concentrations with a maximal increase at 10(-10) mol/L (133+/-34%; p < 0.001). The in vivo short-term effects of bisphosphonates on the formation of early osteoblast precursors were also studied in bone marrow cultures from young female mice treated with weekly subcutaneous injections of etidronate (0.3, 3, and 30 mg/kg) or alendronate (0.3, 3, and 30 microg/kg) and from aging female mice treated with the two lowest doses of both drugs. After 1 month of treatment, etidronate (0.3 and 3 mg/kg) and alendronate (0.3 and 3 microg/kg) significantly increased the number of CFU-F colonies in the bone marrow from young and old animals, whereas the highest dose of both drugs had no effect in young mice. Our results, together with previously reported observations of bone-forming effects in osteoporosis, suggest that bisphosphonates may have, in vivo, a potentially relevant influence on cells of the osteoblastic lineage, distinct from their inhibitory action on osteoclasts.     

Mechanism of the synergistic induction of CYP2H by isopentanol plus ethanol: comparison to glutethimide and relation to induction of 5-aminolevulinate synthase

Mice homozygous for the osteopetrosis (op) mutation are characterized by defective differentiation of osteoclasts, monocytes, and tissue macrophages due to a lack of functional macrophage colony-stimulating factor (M-CSF/CSF-1) activity. In young (4-6 week-old) op/op mice, the bone marrow cavities were filled with spongious bone. In aged (50-72 week-old) op/op mice, the bone marrow cavities were markedly reconstructed and marrow hematopoiesis was expanded. Numbers of osteoclasts and bone marrow macrophages in aged op/op mice were increased but most of the osteoclasts were mononuclear cells and showed poorly developed ruffled borders. Lysosomes of bone marrow macrophages were laden with abundant crystalloid materials in aged op/op mice and aged littermate mice. However, such macrophages were not observed in young op/op mice nor in young littermates. In contrast to the marked increase in numbers of osteoclasts and macrophages in the bone marrow, the number of Kupffer cells in the liver did not increase in aged op/op mice. Kupffer cells in aged op/op mice did not show ultrastructural maturation with aging and contained a few crystalloid structures. M-CSF administration to aged op/op mice induced numerical increases in Kupffer cells and lysosomes in Kupffer cells, disappearance of crystalloid structures in lysosomes of Kupffer cells, and the development of ruffled border in osteoclasts. These findings indicate that M-CSF-independent mechanisms for macrophage and osteoclast development in aged op/op mice are restricted to bone marrow. M-CSF plays important roles in the differentiation of macrophage and osteoclast and the production and function of lysosomes.     

Lymphomagenesis in AKR.Fv-1b congenic mice

In the AKR.Fv-1b congenic strain the Fv-1n allele of the AKR/J mice was substituted with the Fv-1b allele, thereby limiting viral replication and spread of the endogenous N-tropic murine leukemia virus. As a result of this genetic change AKR.Fv-1b mice develop a low spontaneous incidence (7%) of T-cell lymphomas and about 28% of Ly-1+ B-cell lymphomas are observed in old mice. Characteristic changes in thymus subpopulations of AKR/J mice (related to the formation of the dual tropic mink cell focus inducing (MCF) type virus in the thymus) were not observed in the thymus of AKR.Fv-1b mice. In contrast to the low susceptibility to spontaneous T-cell lymphoma development, these mice were highly sensitive to fractionated irradiation or to radiation leukemia virus (a mixture of N- and B-tropic viruses) induced T-cell lymphoma. Potential lymphoma cells (that would ultimately develop into Ly-1+ B-cell lymphomas) were demonstrated in bone marrow and spleens of 16-24-month-old mice. Analysis of the Ly-1+ IgM+ B-cell population in spleens of 18-month-old mice revealed a significant increase in this population (35% versus 2% in young spleens). The spontaneous Ly-1+ B-cell lymphoma incidence could be enhanced (up to 77%) by in vivo administration of anti-CD8 monoclonal antibody or IL-4 to 18-month-old mice. Virological analysis of T/B-cell lymphomas for class I MCF viruses indicated that Class I MCF development was tightly correlated with T-lymphoma development (except radiation induced tumors that showed no MCF provirus involvement). In contrast, Ly-1+ B-cell lymphoma development was independent of Class I MCF pathogenic virus involvement.     

Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor

Bone maintenance requires a continuous source of osteoblasts throughout life. Its remodeling and regeneration during fracture repair is ensured by osteoprogenitor stem cells which are part of the stroma of the bone marrow (BM). Many investigators have reported that in cultured BM stromal cells there is a cell population that will differentiate along an osteogenic lineage if stimulated by the addition of osteogenic inducers, such as dexamethasone (dex), beta-glycerophosphate (beta-GP), transforming growth factor beta-1 (TGF-beta 1) and bone morphogenetic protein-2 (BMP-2). Here we report the effects of demineralized bone matrix (DBM) on the osteogenic differentiation of BM stromal cells in vitro, using morphological criteria, alkaline phosphatase (AP) activity, and calcium accumulation. DBM and DBM-conditioned medium (DBMcm) enhanced bone formation in the presence of dex and beta-GP, whereas DBM particles caused changes in the cell phenotype. Temporal expression of total and skeletal AP by BM stromal cells from 4-week-old rats showed a biphasic pattern enhanced by DBM and suggesting the presence of two cell populations. In one population, AP synthesis reaches a maximum during the first week in culture, following which cells either die or loose their ability to synthesize AP. A second, less abundant population begins to proliferate and synthesize AP during the second and third weeks. The synthesis of AP, which often decreases by the third week, can be maintained at high levels only if DBM is added to the cultures. BM stromal cells isolated from 24- and 48-week-old rats showed a decrease or loss of this biphasic AP expression pattern compared with cells isolated from 4-week-old rats. The addition of DBM to cultures derived from 24- and 48-week-old rats stimulated mostly the second cell population to synthesize AP, suggesting that DBM contains a factor(s) that acts on a specific bone marrow cell population by increasing the proliferation of active cells or inducing the differentiation of dormant cells.     

Learning people''s names following severe closed-head injury

Endothelin-1 (ET-1) was first found as a vasoconstrictor protein excreted by vascular endothelial cells, but recently ET-1 has been considered to have widespread functions that include regulation of osteochondrogenic metabolism. We analyzed sections of head regions in ET-1 knockout mice that are known to have abnormalities in pharyngeal arch-derived tissues and found that there was severe hypoplasia in facial bones. The hypoplasia suggests that the matrix mineralization system of facial bones is disrupted in ET-1-/- homozygous mice. To elucidate whether osteogenic cells in facial bones are the targets for ET-1 and whether expression of bone matrix genes are modulated by ET-1, we examined gene expression of ET-1 receptors, ETA and ETB, and that of the bone matrix proteins, osteonectin (ON) and osteopontin (OP), both in the head regions of ET-1+/- heterozygous and ET-1-/- homozygous mice by means of in situ hybridization. Different patterns of expression between ETA and ETB mRNAs were observed in both groups. In 18.5 days post coitus fetuses, ETA mRNA was most strongly expressed in osteogenic cells along craniofacial bones, but ETB mRNA was most strongly expressed in trunks of trigeminal nerve. This finding suggests that ET-1 may modulate osteogenic cells through ETA receptor but not through ETB receptor. The expression patterns of ETA, OP, and ON mRNAs were distinct between the two groups. In the lower jaw of ET-1+/- heterozygous mice, the ETA, ON, and OP mRNA positive cells were scattered in the inner and outer regions of the thick bone matrix, but in ET-1-/- homozygous mice, cells containing those mRNAs were located close to each other at the surface of thin bone matrix. However, cellular expression of ON and OP mRNAs in osteogenic cells of ET-1-/- homozygous mice was not suppressed as compared with ET-1+/- heterozygous mice. We conclude that ET-1 may regulate proliferation and migration of osteogenic cells in the maxillofacial region, rather than modulating the expression level of ON and OP mRNAs.     

Interleukin-2 inhibits graft-versus-host disease-promoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects

We have recently shown that a short course of high-dose interleukin-2 (IL-2) can markedly inhibit the graft-versus-host disease (GVHD)-promoting activity of donor CD4+ T cells. The difficulty in dissociating GVHD-promoting from graft-versus-leukemia (GVL) effects of alloreactive donor T cells currently prevents clinical bone marrow transplantation (BMT) from fulfilling its full potential. To test the capacity of IL-2 treatment to promote such a dissociation, we have developed a new murine transplantable acute myelogenous leukemia model using a class II major histocompatibility complex-positive BALB/c Moloney murine leukemia virus-induced promonocytic leukemia, 2B-4-2. BALB/c mice receiving 2.5 x 10(5) 2B-4-2 cells intravenously 1 week before irradiation and syngeneic BMT died from leukemia within 2 to 4 weeks after BMT. Administration of syngeneic spleen cells and/or a 2.5-day course of IL-2 treatment alone did not inhibit leukemic mortality. In contrast, administration of non-T-cell-depleted fully allogeneic B10 (H-2b) spleen cells and T-cell-depleted B10 marrow led to a significant delay in leukemic mortality in IL-2-treated mice. In these animals GVHD was inhibited by IL-2 treatment. GVL effects were mediated entirely by donor CD4+ and CD8+ T cells. Remarkably, IL-2 administration did not diminish the magnitude of the GVL effect of either T-cell subset. This was surprising, because CD4-mediated GVHD was inhibited in the same animals in which CD4-mediated GVL effects were not reduced by IL-2 treatment. These results suggest a novel mechanism by which GVHD and GVL effects of a single unprimed alloreactive T-cell subset can be dissociated; different CD4 activities promote GVHD and GVL effects, and the former, but not the latter activities are inhibited by treatment with IL-2.     

Abrogation of TGF-beta activity during retroviral transduction improves murine hematopoietic progenitor and repopulating cell gene transfer efficiency

Transforming growth factor-beta has complex activities on hematopoietic cells. We have previously shown that murine long-term repopulating activity is compromised by ex vivo culture in TGF-beta 1 and conversely is increased by abrogating endogenous TGF-beta activity with a neutralizing antibody. In the current study, we investigated the effect of abrogation of autocrine or paracrine TGF-beta present during retroviral transduction on gene transfer efficiency to primitive hematopoietic cells. Murine marrow cells were cultured and retrovirally transduced for 4 days in the presence of interleukin-3, interleukin-6 and stem cell factor, and either a neutralizing anti-TGF-beta antibody or an isotype control. Committed progenitor cells were analyzed for gene transfer efficiency, and cells were also injected into W/Wv recipient mice for analysis of transduction of long-term repopulating cells. The progenitor (CFU-C) transduction efficiency in the presence of anti-TGF-beta was significantly greater. Semiquantitative PCR analysis and Southern blot analysis for the retroviral marker gene in the blood and bone marrow of recipient mice revealed a significant increase in the transduction efficiency of long-term repopulating cells after culture and transduction in the presence of the anti-TGF-beta. Thus neutralization of TGF-beta activity during retroviral transduction allows more efficient gene transfer into primitive murine hematopoietic cells and may prove beneficial in future clinical gene transfer or therapy trials.     

The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro

Effects of dexamethasone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in cultures of adult human marrow stromal cells. In primary culture, dexamethasone (10(-8) M) increased the number of fibroblast colonies formed but decreased their average size. The number of colonies expressing alkaline phosphatase activity was increased, consistent with the enhancement of osteogenic differentiation by this glucocorticoid. In secondary culture, osteogenic differentiation was assessed by measurement of the steady-state levels of particular mRNAs that are characteristic of cells of the osteoblast lineage. The mRNAs for alpha 1(I)-procollagen, alkaline phosphatase, osteopontin and bone sialoprotein were expressed under all culture conditions used. In contrast, osteocalcin mRNA expression was detectable only in cultures treated with 1,25(OH)2D3 (10(-8) M). Addition of 1,25(OH)2D3 to control increased the expression of the mRNAs for alkaline phosphatase and osteopontin but had no significant effect on bone sialoprotein expression. The highest levels of expression of the mRNAs for alkaline phosphatase, bone sialoprotein and osteocalcin were observed in dexamethasone-treated cultures to which 1,25(OH)2D3 had been added. These results demonstrate that, as earlier found in other species, dexamethasone and 1,25(OH)2D3 promote the osteogenic differentiation of human marrow stromal cells as measured by expression of these osteogenic markers.     

Risk stratifying patients who survive an acute myocardial infarction

While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (approximately 4,000/nucleus) of androgen receptors (AR). Treatment with 5alpha-dihydrotestosterone (5alpha-DHT) inhibited the proliferation of hFOB/AR-6 cells in a dose-dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (<200/nucleus). In hFOB/AR-6 cells, co-treatment with the specific AR antagonist, hydroxyflutamide abolished 5alpha-DHT-induced growth inhibition. Steady-state levels of transforming growth factor-beta1 (TGF-beta1) and TGF-beta-induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR-6 cells with 5alpha-DHT, suggesting a role for the TGF-beta1-TIEG pathway in mediating 5alpha-DHT-induced growth inhibition of hFOB/AR-6 cells. In support of this, co-treatment of hFOB/AR-6 cells with TGF-beta1 (40 pg/ml) reversed the 5alpha-DHT-induced growth inhibition, whereas TGF-beta1 alone at this dose had no effect on hFOB/AR-6 cell proliferation. Furthermore, treatment of hFOB/AR-6 cells with 5alpha-DHT and testosterone (10(-8) M) inhibited basal and 1,25-(OH)2D3-induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast.     

Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro

A number of ultraviolet light-induced tumors that do not grow progressively in 3- or 9-month-old animals grow progressively in animals starting at approximately 15 months of age. We investigated the immune response to one such tumor, 6130, in young and old mice to determine in nature of this breakdown in immune protection with age. The 6130 tumor cells that grow progressively in 22-month-old animals still retain sensitivity of cytotoxic T lymphocytes (CTLs) generated against the highly immunogenic "6130-A" antigen and consequently are still immunologically rejected when transplanted into 3- and 9-month-old animals, but not in mice older than 15 months. Investigation of the CD8+ CTL response found that in animals 3-12 months old, primary and secondary tumor-specific CTL responses to 6130 can be generated in vivo and in vitro. On the other hand, the CD8+ CTL responses to 6130 seen in animals 15 months of age or older were significantly depressed or absent. These results indicate that a decline in antigen-specific CD8+ CTL is the major cause of increased susceptibility to 6130 tumor growth in older animals.     

Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro

We have used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of the osteopontin (Opn, or Spp1, for secreted phosphoprotein 1) gene. Mice homozygous for this disruption fail to express osteopontin (OPN) as assessed at both the mRNA and protein level, although an N-terminal fragment of OPN is detectable at extremely low levels in the bones of -/- animals. The Opn -/- mice are fertile, their litter size is normal, and they develop normally. The bones and teeth of animals not expressing OPN are morphologically normal at the level of light and electron microscopy, and the skeletal structure of young animals is normal as assessed by radiography. Ultrastructurally, proteinaceous structures normally rich in OPN, such as cement lines, persist in the bones of the Opn-/- animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild-type mice. Spleen cells from Opn-/- mice cells formed osteoclasts 3- to 13-fold more frequently than did control Opn+/+ cells, while the extent of osteoclast development from Opn -/- bone marrow cells was about 2- to 4-fold more than from the corresponding wild-type cells. Osteoclast development occurred when Opn-/- spleen cells were differentiated in the presence of Opn-/-osteoblasts, indicating that endogenous OPN is not required for this process. These results suggest that OPN is not essential for normal mouse development and osteogenesis, but can modulate osteoclast differentiation.     

Enhanced acceptance of regenerating bone marrow of random bred newborn mice by random bred adult mice

Regenerating bone marrow of newborn random bred Sabra mice (9-13 days old) was obtained by the administration of two consecutive i.p. injections of hydroxyurea (HU) (2 x 100 mg/kg body wt), three days prior to collection of the marrow cells. The bone marrow of HU-treated newborn mice was assayed for CFU-S, CFU-C and plasma-clot-diffusion-chamber (PCDC) progenitor cells. A fourfold content of CFU-S was found in the regenerating bone marrow compared with that of the control marrow, while the level of CFU-C and PCDC progenitor cells was the same in treated and untreated newborn mice. In lethally irradiated adult, random bred Sabra recipient mice, transfused with regenerating bone marrow from newborn mice, the initial survival rate was greater than in irradiated animals receiving normal newborn marrow (75% as against 50%); marrow repopulation, 10-14 days after transfusion, was also greater in the former than in the latter group of animals (1.5-2x10(6) nucleated cells per femur as compared with 08.-2x10(5)). The bone marrow of these groups of mice was assayed for CFU-S, CFU-C and PCDC progenitor cells; with a cell inoculum of 5 x 10(4) i.v., 10(5) in vitro and 5 x 10(4) per DC, respectively, pluripotent and committed stem cells were detected in the experimental group and were lacking in control recipients. Regenerating bone marrow of newborn mice was also transfused into lethally irradiated splenectomized recipients. In this experimental group there was high mortality, low marrow repopulation and lack of CFU-S (5-10 x 10(4) cell inoculum). The results of this study indicate that, despite genetic differences among random bred Sabra mice, regenerating bone marrow of newborn mice "takes better" than normal marrow in lethally irradiated recipients. Improved marrow acceptance is possibly due to the increased content of activated CFU-S and/or pre-CFU-S in the regenerating bone marrow     

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.     

Vascular pericytes express osteogenic potential in vitro and in vivo

At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three-dimensional nodule-like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage-specific expression of markers of primitive clonogenic marrow stromal cells (STRO-1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.     

EblacZ tumor dormancy in bone marrow and lymph nodes: active control of proliferating tumor cells by CD8+ immune T cells

A well-defined lacZ gene tagged DBA/2 lymphoma (EblacZ) was used to examine the role of host immune responses in controlling tumor dissemination and persistence, as well as metastasis. In s.c. and intra-ear pinna-inoculated mice, low numbers of EblacZ cells homed to the bone marrow and lymph nodes. The frequency of bone marrow-residing tumor cells did not change with the growth of primary tumor or with multiple inoculations of tumor cells. The bone marrow-residing tumor cells expressed the proliferation-associated Ki67 antigen and expanded upon CD8+ depletion. In contrast, inoculation of nu/nu or severe combined immunodeficiency mice or of immune-suppressed DBA/2 mice led to the rapid outgrowth of EblacZ cells in the bone marrow and their metastasis to other organs. Transfer of bone marrow from EblacZ immunized MHC congenic or syngeneic DBA/2 donors, but not from naive donors, protected s.c.-inoculated DBA/2 mice. Protection was abrogated by in vitro depletion of CD8+ T cells prior to transfer of bone marrow. These experiments show that bone marrow and lymph nodes are privileged sites where potentially lethal tumor cells are controlled in a dormant state by the immune system. Metastasis may be a consequence of the breakdown of this immune control.     

Direct visualization of individual DNA molecules by fluorescence microscopy: characterization of the factors affecting signal/background and optimization of imaging conditions using YOYO

The cellular mechanisms involved in osteoblast function and bone formation alterations in osteoporosis have been partly elucidated. Recent studies have shown that bone formation abnormalities in various forms of osteopenia result mainly from defective recruitment of osteoblastic cells. These abnormalities in osteoblast function and bone formation are associated with alterations in the expression or production of several growth factors, such as IGFs and TGF-beta, which modulate the proliferation and activity of bone-forming cells. Bone loss related to aging or unloading is characterized by diminished osteoblast proliferation and reduced local concentrations of IGFs and TGF beta. In contrast, estrogen deficiency increases osteoblast proliferation and IGF-I production. These data suggest that alterations in the production of and/or in cell responsiveness to local growth factors may contribute to the bone formation abnormalities seen in these osteopenic disorders. This suggests that preventive or curative treatment with growth factors may be beneficial in osteopenia due predominantly to decreased bone formation. Low doses of IGF-I or TGF-beta have been reported to increase osteoblast recruitment and differentiation, leading to enhanced trabecular bone formation and decreased bone loss in models of osteopenia induced by aging, estrogen deficiency and unloading. A few clinical trials also suggest that low doses of growth factors may stimulate bone formation. Although these findings open up new prospects for the prevention and treatment of osteopenic disorders, progress in this direction awaits the development of factors or analogs that are capable of locally and specifically increasing osteoblast recruitment and differentiation without including side-effects.