Linear Regression as a Method to Prioritize Humanitarian Efforts in Stability Operations.

With a mandate to work by, with, and through host nation partners, Special Operations Forces (SOF) often face the challenge of pursuing humanitarian endeavors in the hopes of securing access to a specific population and mapping the human terrain. Likewise, should limitations in the rules of engagement (ROE) shift incentives from lethal to non-lethal effects, commanders must find unique ways to exert influence on the operational environment. However, with inevitable resource constraints such as money and time, it can be exceedingly difficult to determine which humanitarian project to undertake, especially in a population whose needs are multifaceted. Linear regression, a statistical tool available within the standard Microsoft Excel package on government computers, permits the modeling of predictive outcomes between a number of independent variables against a dependent variable. This allows the determination of significance and effect for each independent variable, which can facilitate a thoughtful recommendation to commanders for project selection. Using Iraq as an example, publicly available information (PAI) provides a wealth of records to make data-driven assessments for mutually beneficial shaping efforts in a stability operations framework. Additionally, this paper will highlight how data can be analyzed without a reliance on statistical software that is unlikely to be present in the tactical environment.

CD55 deficiency protects against atherosclerosis in ApoE-deficient mice via C3a modulation of lipid metabolism.

Atherosclerosis, the leading cause of death in the Western world, is driven by chronic inflammation within the artery wall. Elements of the complement cascade are implicated in the pathogenesis, because complement proteins and their activation products are found in the atherosclerotic plaque. We examined the role of CD55, a membrane inhibitor of the complement component 3 (C3) convertase, which converts C3 into C3a and C3b, in atherosclerosis. CD55-deficient (CD55(-/-)) mice were crossed onto the atherosclerosis-prone apolipoprotein E (apoE)-deficient (apoE(-/-)) background. High fat-fed male apoE(-/-)/CD55(-/-) mice were strongly protected from developing atherosclerosis compared with apoE(-/-) controls. Lipid profiling showed significantly lower levels of triglycerides, nonesterified fatty acids, and cholesterol in apoE(-/-)/CD55(-/-) mice than that in controls after high-fat feeding, whereas body fat in apoE(-/-)/CD55(-/-) mice content was increased. Plasma levels of C3 fell, whereas concentrations of C3adesArg (alias acylation stimulating protein; ASP), produced by serum carboxypeptidase N-mediated desargination of C3a, increased in nonfasted high fat-fed apoE(-/-)/CD55(-/-) mice, indicating complement activation. Thus, complement dysregulation in the absence of CD55 provoked increased C3adesArg production that, in turn, caused altered lipid handling, resulting in atheroprotection and increased adiposity. Interventions that target complement activation in adipose tissue should be explored as lipid-decreasing strategies.

The influence of leptin on trabecular architecture and marrow adiposity in GH-deficient rats.

The relationship between the degree of GH deficiency and impaired bone integrity is not simple and may be influenced by related endocrine variables. To test the hypothesis that elevated adiposity and hyperleptinaemia are contributory factors, we quantified femoral trabecular organisation in two models of GH deficiency with divergent degrees of adiposity - the moderately GH-deficient/hyperleptinaemic transgenic growth retarded (Tgr) rat and the profoundly GH-deficient/hypoleptinaemic dw/dw rat. Trabecular density (bone volume/total volume) and surface were reduced by 16% in dw/dw males, with a more fragmented trabecular lattice. This impairment was more pronounced in Tgr rats, with trabecular number and density further reduced (by an additional 21%) and relative surface (bone surface/bone volume), trabecular convexity (structural modal index) and fragmentation (pattern factor) increased. To establish whether the presence of obesity/hyperleptinaemia exacerbates bone impairment in GH deficiency, trabecular structure was assessed in dw/dw rats following diet-induced obesity (DIO). DIO had minimal effect on trabecular architecture, the increased concavity of trabecular surfaces being the only observable effect. Similarly, infusion of leptin into the tibial bone marrow cavity had no effect on trabecular organisation or tibial growth in wild-type rats. However, while this procedure also failed to affect trabecular architecture or osteoclast number in dw/dw rats, distal osteoblast surface was increased by 23%, marrow adipocyte number and epiphyseal plate width being reduced (by 40 and 5% respectively), without increasing caspase-3 immunoreactivity. These findings suggest that while leptin may directly inhibit adipocyte differentiation and favour osteoblast production, hyperleptinaemia makes only a minimal contribution to the impairment of bone structure in GH deficiency.

Comparison of bone mineral density in medial coronoid processes of dogs with and without medial coronoid process fragmentation.

OBJECTIVE: To quantify bone mineral density (BMD) in the medial coronoid process (MCP) of dogs with and without fragmented medial coronoid processes (FMCPs) by use of dualenergy x-ray absorptiometry. SAMPLE POPULATION: 50 osteochondral samples from 31 dogs that underwent subtotal coronoid ostectomy for unilateral or bilateral FMCP and 10 control osteochondral samples of the MCP collected from forelimbs of 5 cadaveric Greyhounds. PROCEDURES: Each sample was mounted in proximodistal and mediolateral orientations for BMD determinations via dual-energy x-ray absorptiometry, and area-of-interest data (0.03-cm(2) increments) were obtained. Values of BMD were compared between left and right limb control samples, between control and FMCP samples, and between axial and abaxial regions of the control or FMCP samples. RESULTS: The BMD in control and FMCP samples in both proximodistal and mediolateral orientations differed significantly. Mean BMD throughout the MCP was decreased in FMCP samples, compared with control sample findings. In both control and FMCP samples, BMD of the abaxial half of the MCP was 50% higher than that of the axial portion. CONCLUSIONS AND CLINICAL RELEVANCE: The similar pattern of BMD in osteochondral samples of the MCP in dogs with and without FMCP indicated that the MCP was eccentrically loaded during weight bearing. Topographic variation in BMD in the MCP, and hence tolerance to compressive loading, suggested that the abaxial portion of the MCP in dogs was more resistant to compressive load than was the axial edge. This difference may predispose the coronoid process to microcrack formation and fragmentation at that juxtaposition.

Ultrasound mimics the effect of mechanical loading on bone formation in vivo on rat ulnae.

While the effect of ultrasound as an extreme example of low-magnitude high-frequency stimulation has been explored in the response of bone to injury, little is known about its effect on normal bone. This experiment was designed to test the hypothesis that ultrasound exerts a similar influence on bone as mechanical stimulation at a physiological level. Three groups of female Wistar rats were anaesthetised (6 per group). In one group, the left ulna was loaded cyclically in vivo 40 times, repeated on a further 5 occasions on alternate days. In a second group, transcutaneous low-intensity pulsed ultrasound stimulation was applied to the left ulnae for the same duration as the period of loading. In a third group, loading and ultrasound stimulation were applied concurrently. The right ulna served as non-loaded control in each animal. At the end of the experiment after 14 days, both ulnae were removed. Induced bone formation was assessed by measuring the proportion of medial periosteal bone surface with double label (dLS/BS, %) and by calculation of mineral apposition rate (MAR) from the inter-label distance. All three treatments induced a significant periosteal response, increasing dLS/BS values from <10% in control limbs to >80% in treated limbs. Increases in MAR of experimental ulnae versus contralateral control ulnae were 2.9 (+/-0.9), 8.6 (+/-2.4) and 8.7 microm (+/-3.2) for the ultrasound only, ultrasound and load, and load only groups, respectively. The effects of loading plus ultrasound were not significantly different from ultrasound alone. These data suggest that ultrasound is able to induce changes in bone that share at least some features with mechanical loading.

An ENU-induced mutation in the Ankrd11 gene results in an osteopenia-like phenotype in the mouse mutant Yoda.

The mechanisms that regulate bone mass are important in a variety of complex diseases such as osteopenia and osteoporosis. Regulation of bone mass is a polygenic trait and is also influenced by various environmental and lifestyle factors, making analysis of the genetic basis difficult. As an effort toward identifying novel genes involved in regulation of bone mass, N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has been utilized. Here we describe a mouse mutant termed Yoda that was identified in an ENU mutagenesis screen for dominantly acting mutations. Mice heterozygous for the Yoda mutation exhibit craniofacial abnormalities: shortened snouts, wider skulls, and deformed nasal bones, underlined by altered morphology of frontonasal sutures and failure of interfrontal suture to close. A major feature of the mutant is reduced bone mineral density. Homozygosity for the mutation results in embryonic lethality. Positional cloning of the locus identified a missense mutation in a highly conserved region of the ankyrin repeat domain 11 gene (Ankrd11). This gene has not been previously associated with bone metabolism and, thus, identifies a novel genetic regulator of bone homeostasis.

Impaired growth plate function in bmp-6 null mice.

Bone morphogenetic protein 6 (BMP-6) is expressed by different skeletal cells including osteoblasts and growth plate chondrocytes, suggesting roles in bone formation and growth regulation. To address these possibilities, we examined whether cancellous and cortical bone parameters, or indices of growth plate function, are altered in bmp-6 null mice as assessed under basal conditions, and following stimulation of bone formation and suppression of growth by estrogen treatment. Ten-week-old female littermate bmp-6 null and wild-type (WT) mice were administered vehicle or E(2) 4, 40, 400 or 4,000 microg/kg/day by daily sc injection for 28 days (6-8 per group). Tibias were removed, and detailed histomorphometric analysis of the proximal metaphysis and growth plates, and tibial diaphysis were performed on longitudinal and transverse sections respectively. Long bone area as measured by DXA was reduced in vehicle-treated bmp-6 null mice compared with WT littermate controls. In addition, vehicle-treated bmp-6 null mice had a reduced cross-sectional area at the tibial mid-diaphysis as assessed by histomorphometry, whereas cancellous bone indices were unaffected. Histomorphometry of the proximal tibial metaphysis demonstrated a defect in bone formation immediately adjacent to the growth plate in bmp-6 null mice compared to WT mice following E(2) treatment. E(2) administration was also associated with a dose-responsive decrease in longitudinal growth rate, and proliferative and hypertrophic zone parameters of the growth plate (p<0.0001). Significantly greater reductions following E(2) treatment were observed in longitudinal growth rate (p<0.01), proliferating and hypertorphic zone widths (p<0.001), and proliferating (p<0.0002) and hypertrophic (p<0.002) cells per column of bmp-6 null mice compared to WT mice. Our observation that long bones are reduced in size compared to wild-type mice primarily through a decrease in cortical cross-sectional area, whilst cancellous bone mass is unaltered, suggests a non-redundant role for BMP-6 in periosteal but not trabecular bone formation. Moreover, growth plate function was reduced in bmp-6 null mice receiving estrogen, leading to an impaired cancellous bone response to estrogen at the highest dose, suggesting that BMP-6 also plays a physiological role in maintaining growth plate function.

Osteopenia in Sparc (osteonectin)-deficient mice: characterization of phenotypic determinants of femoral strength and changes in gene expression.

Sparc null mutants have been generated independently via targeted mutations in exons 4 and 6. Previous studies have identified low-turnover osteopenia in the 129Sv/C57BL/6 exon 4 knockout. Since both Sparc null mutations result in complete absence of Sparc protein, similar phenotypic outcomes are likely. However, genetic background (strain) and/or linkage disequilibrium effects can influence phenotype. Different inactivating mutations should be tested in various mouse strains; similar phenotypic outcomes can then confidently be assigned to the mutated gene. We have evaluated the bone phenotype in the 129Sv/EvSparc(tm1cam) exon 6 knockout at 4 and 9 mo, using physical measurement, mechanical strength tests, and DXA scanning. We have also quantified bone marrow adiposity and circulating leptin levels to assess adipose tissue metabolism. 129Sv/EvSparc(tm1cam) null mice show decreased bone mineral density and bone mineral content and increased mechanical fragility of bone, in line with previous studies. Differences were also noted. Increased body weight and levels of bone marrow adiposity but decreased circulating leptin concentrations were identified at 4, but not 9 mo, and 129Sv/EvSparc(tm1cam) null mice also had shorter femurs. Molecular phenotyping was carried out using mouse HGMP NIA microarrays with cortical femur samples at various ages, using semiquantitative RT-PCR validation. We identified 429 genes highly expressed in normal bone. Six genes (Sparc, Zfp162, Bysl, E2F4, two ESTs) are differentially regulated in 129Sv/EvSparc(tm1cam) cortical femur vs. 129Sv/Ev controls. We confirm low-turnover osteopenia as a feature of the Sparc null phenotype, identifying the usefulness of this mouse as a model for human osteoporosis.

Mice rendered severely deficient in megakaryocytes through targeted gene deletion of the thrombopoietin receptor c-Mpl have a normal skeletal phenotype.

To explore whether a functional relationship exists between megakaryocytes and the cellular processes responsible for bone formation, we examined if Mpl ( -/- ) mice, which are severely megakaryocyte-deficient through c-Mpl gene deletion, have an abnormal skeletal phenotype compared to Mpl ( +/- ) and wild-type littermates. We also analyzed whether the osteogenic response to high-dose estrogen treatment is altered in Mpl ( -/- ) mice. Megakaryocyte numbers and skeletal indices were compared between Mpl ( -/- ) mice and littermate Mpl ( +/- ) and wild-type 12-week-old mice (six per group). Dual-energy X-ray absorbtiometry of whole body, excised tibias, and femurs was performed. Histomorphometric analyses of the proximal metaphysis and mid-diaphysis were carried out on longitudinal and transverse sections, respectively. Histomorphometry was performed on the proximal tibial metaphysis of four Mpl ( -/- ) and four wild-type mice following high-dose estrogen treatment (0.5 mg/animal/week) for 4 weeks. Mpl ( -/- ) mice had 10% the megakaryocyte number of Mpl ( +/- ) and wild-type littermates. Bone mineral density values in Mpl ( -/- ) mice were identical to those in Mpl ( +/- ) and wild-type mice for whole body, femur, and tibia. Histomorphometric analysis demonstrated that cancellous and cortical tibial bone parameters were similar across all genotypes. The osteogenic response to estrogen treatment was indistinguishable between Mpl ( -/- )and wild-type mice. We found that mice severely deficient in megakaryocytes have a normal skeletal phenotype. Additionally, the deficiency did not diminish the osteogenic marrow response to high-dose estrogen treatment. These results represent the first in vivo evidence that severe megakaryocyte deficiency does not affect bone formation, suggesting that this process is not dependent on normal megakaryocyte number.

Pharmacological regulation of adult stem cells: chondrogenesis can be induced using a synthetic inhibitor of the retinoic acid receptor.

Conventional methods for regulating the differentiation of stem cells are largely based on the use of biological agents such as growth factors. We hypothesize that stem cell differentiation could be driven by specific synthetic molecules. If true, this would offer the possibility of screening chemical libraries to develop pharmacological agents with improved efficacy. To test our hypothesis, we have determined which, if any, of the nuclear receptor superfamily might be involved in chondrogenesis. We used fluorescence-activated cell sorting, as well as quantitative polymerase chain reaction, to study expression of a range of nuclear receptors in the undifferentiated mesenchymal population and after growth factor-driven differentiation of these cells to chondrocytes. In this way, we identified retinoic acid receptor beta (RAR beta) as a potential pharmacological target. A low molecular weight synthetic inhibitor of the RAR alpha and RAR beta receptors was able to induce chondrogenic differentiation of mesenchymal stem cells derived from osteoarthritis patients, in the absence of serum and growth factors. Furthermore, the pathway is independent of SOX9 upregulation and does not lead to hypertrophy. When mesenchymal cells were seeded on to polyglycolic acid scaffolds and cultured with LE135, there was a dose-dependent formation of cartilage, demonstrated both histologically and by biochemical analysis of the collagen component of the extracellular matrix. These results demonstrate the feasibility of a pharmacological approach to the regulation of stem cell function. Disclosure of potential conflicts of interest is found at the end of this article.

High-dose estrogen-induced osteogenesis is decreased in aged RUNX2(+/-) mice.

Runx2 is a transcription factor that is not only critical in embryonic skeletal development but also important in regulating osteoblast function in the adult. Heterozygosity of RUNX2 (RUNX2(+/-)) leads to haploinsufficiency and manifests as a condition with distinctive skeletal features in humans and mice. Aged but not young RUNX2(+/-) adult mice may also display reduced intramembranous bone formation. To clarify the role of Runx2 in intramembranous bone formation in adult mice a histomorphometric study was performed to compare the osteogenic response to high-dose estrogen in RUNX2(+/-) and wild-type mice. Young (10 weeks) and aged (26 weeks) RUNX2(+/-) and wild-type littermate mice were treated with vehicle or high-dose estrogen (0.5 mg/animal/week) by subcutaneous injection for 4 weeks. Mice were divided into 8 groups according to age, genotype and treatment with 6 animals per group. Following sacrifice, longitudinal tibial sections were prepared and examined by static and dynamic histomorphometry. Estrogen treatment induced formation of new cancellous bone in both wild-type and RUNX2(+/-) mice. This occurred to the same extent in young mice of both genotypes. However, in the aged RUNX2(+/-) mice this response as assessed by bone volume (BV/TV%) was decreased by over 70% (p<0.001) when compared to aged wild-type mice. Furthermore, significant reductions in cancellous double-labelled surfaces (dls/TV, 1.7+/-0.2 vs 1.0+/-0.4 mm(2)/mm(3), p<0.05) and mineral apposition rate (1.8+/-0.1 vs 1.4+/-0.1 microm/day, p<0.01) were observed in aged RUNX2(+/-) mice compared to wild-types. Aged RUNX2(+/-) mice display an abrogated osteogenic response to high-dose estrogen. This may have occurred through combined reductions in recruitment of osteoprogenitor cells, osteoblast activity and mineralization. Since the characteristic histological changes in the marrow cavity which precede the formation of cancellous bone following estrogen treatment was seen in the aged RUNX2(+/-) mice we suggest that they may eventually be capable of a full osteogenic response but haploinsufficiency leads to delayed bone formation.

Effects on osteoclast and osteoblast activities in cultured mouse calvarial bones by synovial fluids from patients with a loose joint prosthesis and from osteoarthritis patients.

Aseptic loosening of a joint prosthesis is associated with remodelling of bone tissue in the vicinity of the prosthesis. In the present study, we investigated the effects of synovial fluid (SF) from patients with a loose prosthetic component and periprosthetic osteolysis on osteoclast and osteoblast activities in vitro and made comparisons with the effects of SF from patients with osteoarthritis (OA). Bone resorption was assessed by the release of calcium 45 (45Ca) from cultured calvariae. The mRNA expression in calvarial bones of molecules known to be involved in osteoclast and osteoblast differentiation was assessed using semi-quantitative reverse transcription-polymerase chain reaction (PCR) and real-time PCR. SFs from patients with a loose joint prosthesis and patients with OA, but not SFs from healthy subjects, significantly enhanced 45Ca release, effects associated with increased mRNA expression of calcitonin receptor and tartrate-resistant acid phosphatase. The mRNA expression of receptor activator of nuclear factor-kappa-B ligand (rankl) and osteoprotegerin (opg) was enhanced by SFs from both patient categories. The mRNA expressions of nfat2 (nuclear factor of activated T cells 2) and oscar (osteoclast-associated receptor) were enhanced only by SFs from patients with OA, whereas the mRNA expressions of dap12 (DNAX-activating protein 12) and fcrgamma (Fc receptor common gamma subunit) were not affected by either of the two SF types. Bone resorption induced by SFs was inhibited by addition of OPG. Antibodies neutralising interleukin (IL)-1alpha, IL-1beta, soluble IL-6 receptor, IL-17, or tumour necrosis factor-alpha, when added to individual SFs, only occasionally decreased the bone-resorbing activity. The mRNA expression of alkaline phosphatase and osteocalcin was increased by SFs from patients with OA, whereas only osteocalcin mRNA was increased by SFs from patients with a loose prosthesis. Our findings demonstrate the presence of a factor (or factors) stimulating both osteoclast and osteoblast activities in SFs from patients with a loose joint prosthesis and periprosthetic osteolysis as well as in SFs from patients with OA. SF-induced bone resorption was dependent on activation of the RANKL/RANK/OPG pathway. The bone-resorbing activity could not be attributed solely to any of the known pro-inflammatory cytokines, well known to stimulate bone resorption, or to RANKL or prostaglandin E2 in SFs. The data indicate that SFs from patients with a loose prosthesis or with OA stimulate bone resorption and that SFs from patients with OA are more prone to enhance bone formation.

Isolation of C15: a novel antibody generated by phage display against mesenchymal stem cell-enriched fractions of adult human marrow.

Adult bone marrow stroma contains a source of mesenchymal stem cells (MSC) that have the capacity to self-renew and differentiate into multiple stromal lineages. These rare cells can be visualised indirectly by the formation of heterogeneous colonies, containing stem cells and their differentiated progeny in long-term culture. If MSC and their associated progenitor and precursor populations are to reach their full therapeutic potential, markers will be required to identify and characterize specific bone marrow stromal subsets. We sought to use phage display to generate antibodies against bone marrow mononuclear cells (BMMNC) enriched for colony forming cells. Initially, we identified our target cell population by comparing the colony forming efficiency (CFE) of CD49a-positive, STRO-1-positive and CD45-negative BMMNC subpopulations with unseparated BMMNC. Selection with anti-CD49a gave the greatest enrichment (19-fold) of colony forming cells and in light of these findings, we generated phage antibodies against CD49a-positive BMMNC by simultaneous positive/negative selection. A dominant clone (C15), generated after 3 rounds of selection, has been isolated and sequenced, then characterized for cell and tissue specificity. Sequence analysis showed that the V(H) and V(L) gene segments of C15 aligned most closely to the VH26/DP-47 and IGLV3S1/DPL16 germline V segments found in the synthetic repertoire. C15 bound to 4% of freshly isolated BMMNC and localized to osteoblastic cells and proximal marrow cells in areas of active bone formation in sections of osteophyte. C15 binding was upregulated in cultured bone marrow stromal cells (BMSC) and was also detected on bone-derived cell lines. This report demonstrates that phage display is a powerful tool for the isolation of antibodies against rare cell populations, and provides a platform for the future application of this technology in the search for antigens on MSC and other rare cell populations.

Estrogen receptor-alpha dependency of estrogen's stimulatory action on cancellous bone formation in male mice.

We examined whether estrogen receptor (ER)alpha is required for estrogen to stimulate cancellous bone formation in long bones of male mice. 17 beta-Estradiol (E(2)) was administered to ER alpha(-/-) male mice or wild-type (WT) littermate controls at 40, 400, or 4000 microg/kg by daily sc injection for 28 d and histomorphometric analysis performed at the distal femoral metaphysis. In WT mice, treatment with E(2) (40 microg/kg per d) increased the proportion of cancellous bone surfaces undergoing mineralization and stimulated mineral apposition rate. In addition, higher doses of E(2) induced the formation of new cancellous bone formation surfaces in WT mice. In contrast, E(2) had little effect on any of these parameters in ER alpha(-/-) mice. Immunohistochemistry was subsequently performed using an ER alpha-specific C-terminal polyclonal antibody. In WT mice, ER alpha was expressed both by cancellous osteoblasts and a significant proportion of mononuclear bone marrow cells. Immunoreactivity was also observed in cancellous osteoblasts of ER alpha(-/-) mice, resulting from expression of the activation function-1-deficient 46-kDa ER alpha isoform previously reported to be expressed in normal osteoblasts and bones of ER alpha(-/-) mice. Taken together, our results suggest that estrogen stimulates bone formation in mouse long bones via a mechanism that requires the presence of full-length ER alpha possessing activation function-1.

Zoledronic acid treatment of 5T2MM-bearing mice inhibits the development of myeloma bone disease: evidence for decreased osteolysis, tumor burden and angiogenesis, and increased survival.

Multiple myeloma is characterized by the growth of plasma cells in the bone marrow and the development of osteolytic bone disease. Myeloma cells are found closely associated with bone, and targeting this environment may therefore affect both the bone disease and the growth of myeloma cells. We have investigated the effect of the potent bisphosphonate, zoledronic acid, on the development of bone disease, tumor burden, and disease-free survival in the 5T2MM model of myeloma. 5T2MM murine myeloma cells were injected intravenously into C57BL/KaLwRij mice. After 8 weeks, all animals had a paraprotein. Animals were treated with zoledronic acid (120 microg/kg, subcutaneously, twice weekly) or vehicle, from the time of tumor cell injection or from paraprotein detection for 12 or 4 weeks, respectively. All animals injected with tumor cells developed osteolytic lesions, a decrease in cancellous bone volume, an increase in osteoclast perimeter, and a decrease in bone mineral density. Zoledronic acid prevented the formation of lesions, prevented cancellous bone loss and loss of bone mineral density, and reduced osteoclast perimeter. Zoledronic acid also decreased paraprotein concentration, decreased tumor burden, and reduced angiogenesis. In separate experiments, Kaplan-Meier analysis demonstrated a significant increase in survival after treatment with zoledronic acid when compared with control (47 vs. 35 days). A single dose of zoledronic acid was also shown to be effective in preventing the development of osteolytic bone disease. These data show that zoledronic acid is able to prevent the development of osteolytic bone disease, decrease tumor burden in bone, and increase survival in a model of established myeloma.

Estrogen-induced osteogenesis in mice is associated with the appearance of Cbfa1-expressing bone marrow cells.

Cbfa1 is a transcription factor recognised as being involved in early osteoblast differentiation during embryonic skeletogenesis. To determine whether Cbfa1 plays a similar role in bone formation in the adult, we analysed whether its expression is altered during estrogen-induced osteogenesis, following our recent studies which suggest that this response involves the generation of early osteoblast precursors within bone marrow. To facilitate identification of Cbfa1-expressing cells, these studies were performed in mice heterozygous for a cbfa1 gene deletion (cbfa1(+/-)) using beta-galactosidase (lacZ) as a genetic marker. Cbfa1-expressing cells were identified by lacZ staining of longitudinal sections of the proximal tibial metaphysis. Treatment of cbfa1(+/-) mice with 17beta-estradiol 0.5 mg/week for 24 days led to the appearance of new cancellous bone surfaces. This response was associated with a marked increase in number of Cbfa1-expressing cells within the metaphysis, consisting not only of osteoblasts on bone surfaces but also of cells within the adjacent bone marrow. We subsequently enumerated Cbfa1-expressing cells at earlier time-points following estrogen, in sections co-stained for ALP activity. After 4 days of estrogen treatment, a population of cells appeared within the marrow cavity which expressed Cbfa1, but were negative for ALP. At later time-points, large numbers of Cbfa1 + bone marrow cells were still present, but the majority of these were close to new trabecular bone surfaces at sites which showed high levels of ALP activity. An equivalent distribution of Cbfa1-expressing cells was observed in further studies where Cbfa1 expression was analysed in wild-type mice by immunohistochemistry. We conclude that estrogen-induced osteogenesis is associated with the appearance of a population of Cbfa1-expressing cells within bone marrow, which we hypothesize to represent the osteoblast precursor population responsible for subsequent new bone formation.