Wnt7b expressed by hypertrophic chondrocytes is a stimulatory factor for endochondral ossification that is regulated by Smad4 activity.

Endochondral ossification contributes to longitudinal skeletal growth. Osteoblasts, which are bone-forming cells, appear close to terminally differentiated hypertrophic chondrocytes during endochondral ossification. We established mice with conditional knockout (cKO) of Smad4, an essential co-activator for transforming growth factor ß family signaling. The mice showed a marked increase in bone volume in the metaphysis as a result of increased bone formation by osteoblasts, in which ß-catenin, an effector of canonical Wnt signaling, accumulated. We identified Wnt7b as a factor with increased expression in growth plate cartilage in Smad4 cKO mice. Wnt7b mRNA was expressed in differentiated chondrocytes and suppressed by BMP4 stimulation. Ablation of Wnt7b blunted the increase in bone in adult Smad4 cKO mice and reduced skeletal growth in juvenile mice. Overall, we conclude that Wnt7b is a crucial factor secreted from hypertrophic chondrocytes to initiate endochondral ossification. These results suggest that Smad4-dependent BMP signaling regulates the Wnt7b-ß-catenin axis during endochondral ossification.

Critical Roles of NF-κB Signaling Molecules in Bone Metabolism Revealed by Genetic Mutations in Osteopetrosis.

The nuclear factor-κB (NF-κB) transcription factor family consists of five related proteins, RelA (p65), c-Rel, RelB, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). These proteins are important not only for inflammation and the immune response but also for bone metabolism. Activation of NF-κB occurs via the classic and alternative pathways. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, activate the former, and cytokines involved in lymph node formation, such as receptor activator of NF-κB ligand (RANKL) and CD40L, activate the latter. p50 and p52 double-knockout mice revealed severe osteopetrosis due to the total lack of osteoclasts, which are specialized cells for bone resorption. This finding suggests that the activation of NF-κB is required for osteoclast differentiation. The NF-κB signaling pathway is controlled by various regulators, including NF-κB essential modulator (NEMO), which is encoded by the IKBKG gene. In recent years, mutant forms of the IKBKG gene have been reported as causative genes of osteopetrosis, lymphedema, hypohidrotic ectodermal dysplasia, and immunodeficiency (OL-EDA-ID). In addition, a mutation in the RELA gene, encoding RelA, has been reported for the first time in newborns with high neonatal bone mass. Osteopetrosis is characterized by a diffuse increase in bone mass, ranging from a lethal form observed in newborns to an asymptomatic form that appears in adulthood. This review describes the genetic mutations in NF-κB signaling molecules that have been identified in patients with osteopetrosis.

Inhibitory effects of sesquiterpene lactones from the Indonesian marine sponge Lamellodysidea cf. herbacea on bone morphogenetic protein-induced osteoblastic differentiation.

A new unique sesquiterpene lactone, bicyclolamellolactone A (1), was isolated together with two known monocyclofarnesol-type sesquiterpenes, lamellolactones A (2) and B (3), from the Indonesian marine sponge Lamellodysidea sp. (cf. L. herbacea). The planar structure of 1 was assigned based on its spectroscopic data (1D and 2D NMR, HRESIMS, UV, and IR spectra). The relative and absolute configuration of 1 was determined by comparison of its calculated and experimental electronic circular dichroism spectra in combination with NOESY correlations. Compounds 1-3 inhibited bone morphogenic protein (BMP)-induced alkaline phosphatase activity in mutant BMP receptor-carrying C2C12 cells with IC50 values of 51, 4.6, and 20 µM, respectively.

Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva.

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

A Door Opens for Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP), characterized by extra bone formation in soft tissues, is caused by a gain-of-function mutation in ACVR1, a transmembrane receptor. Recently, a potential treatment was developed by identifying a novel molecular mechanism underlying bone formation in FOP. These findings have opened the door to beating FOP.

BMPR2 inhibits activin and BMP signaling via wild-type ALK2.

TGF-ß/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-ß superfamily member, inhibits growth of multiple myeloma cells, but the mechanism for this is unknown. We therefore aimed to clarify how activins affect myeloma cell survival. Activin A activates the transcription factors SMAD2/3 through the ALK4 type 1 receptor, but may also activate SMAD1/5/8 through mutated variants of the type 1 receptor ALK2 (also known as ACVR1). We demonstrate that activin A and B activate SMAD1/5/8 in myeloma cells through endogenous wild-type ALK2. Knockdown of the type 2 receptor BMPR2 strongly potentiated activin A- and activin B-induced activation of SMAD1/5/8 and subsequent cell death. Furthermore, activity of BMP6, BMP7 or BMP9, which may also signal via ALK2, was potentiated by knockdown of BMPR2. Similar results were seen in HepG2 liver carcinoma cells. We propose that BMPR2 inhibits ALK2-mediated signaling by preventing ALK2 from oligomerizing with the type 2 receptors ACVR2A and ACVR2B, which are necessary for activation of ALK2 by activins and several BMPs. In conclusion, BMPR2 could be explored as a possible target for therapy in patients with multiple myeloma.This article has an associated First Person interview with the first author of the paper.

Screening for Small Molecule Inhibitors of BMP-Induced Osteoblastic Differentiation from Indonesian Marine Invertebrates.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder with heterotopic ossification (HO) in soft tissues. The abnormal activation of bone morphogenetic protein (BMP) signaling by a mutant activin receptor-like kinase-2 (ALK2) leads to the development of HO in FOP patients, and, thus, BMP signaling inhibitors are promising therapeutic applications for FOP. In the present study, we screened extracts of 188 Indonesian marine invertebrates for small molecular inhibitors of BMP-induced alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation in a C2C12 cell line stably expressing ALK2(R206H) (C2C12(R206H) cells), and identified five marine sponges with potent ALP inhibitory activities. The activity-guided purification of an EtOH extract of marine sponge Dysidea sp. (No. 256) resulted in the isolation of dysidenin (1), herbasterol (2), and stellettasterol (3) as active components. Compounds 1-3 inhibited ALP activity in C2C12(R206H) cells with IC50 values of 2.3, 4.3, and 4.2 µM, respectively, without any cytotoxicity, even at 18.4-21.4 µM. The direct effects of BMP signaling examined using the Id1WT4F-luciferase reporter assay showed that compounds 1-3 did not decrease the reporter activity, suggesting that they inhibit the downstream of the Smad transcriptional step in BMP signaling.

Fibrodysplasia ossificans progressiva: Review and research activities in Japan.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic skeletal disorder manifesting progressive heterotopic ossification (HO) and congenital malformation of the great toes. Since 2007, we have conducted research on FOP. Here, we review the findings on FOP published to date, including the results of our research. Epidemiological studies in Japan have indicated that FOP has nearly the same prevalence in Japan as in the rest of the world. Basic research on its pathoetiology has progressed rapidly since the identification of the causal gene in 2006. Clinical and radiological findings have been thoroughly researched, including early radiological signs, and diagnostic criteria were established, designating FOP as an intractable disease in Japan. In patients with FOP, the progression of HO is associated with numerous disabilities, often manifesting in vicious cycles that can lead to early mortality. Through cross-sectional and short-term longitudinal studies, we have explored patient education, quality of life, and activities of daily living among Japanese patients. The management of FOP requires education of patients and caregivers, the use of medications to settle inflammation and flare-ups, instructions to ensure proper oral care, and other compensatory approaches that aid in rehabilitation. An avoidance of medical intervention, which may cause HO to progress, is also important. The advent of new drugs to prevent HO could have clinical benefit.

Discovery of Heterotopic Bone-Inducing Activity in Hard Tissues and the TGF-ß Superfamily.

Bone is a unique organ because it can be experimentally induced in soft tissues by implanting a single growth factor, bone morphogenetic protein (BMP). Heterotopic bone-inducing activity was found in demineralized bone matrix in 1965. The characterization of this activity in bone enabled the purification and molecular cloning of BMPs and showed that they are members of the transforming growth factor-ß (TGF-ß) superfamily. Assay systems developed for this bone-inducing activity revealed the molecular mechanisms of the intracellular signaling of members of the superfamily, including BMPs. Moreover, they are being applied to elucidate molecular mechanisms and to develop novel therapeutics for a disease caused by an abnormality in BMP signaling.

[Fibrodysplasia ossificans progressiva and osteoimmunology].

Fibrodysplasia ossificans progressive (FOP) is a genetic disorder characterized by progressive heterotopic ossification (HO) in skeletal muscle, tendons and ligaments. FOP is caused by gain-of-function mutations of ALK2, a receptor of bone morphogenetic proteins. Immune responses have been suggested to be involved in HO in FOP, because muscle trauma induces acute HO in patients with FOP. Recently, Activin A, a non-osteogenic ligand, was identified as a ligand of the mutated ALK2 to induce HO. It was suggested that Activin A is a novel interface between FOP and osteoimmunology.

Involvement of PRIP (Phospholipase C-Related But Catalytically Inactive Protein) in BMP-Induced Smad Signaling in Osteoblast Differentiation.

Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.

Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-ß1 (TGF-ß1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs.

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.

Activation of NF-κB signaling regulates ovariectomy-induced bone loss and weight gain.

Postmenopausal women experience bone loss and weight gain. To date, crosstalk between estrogen receptor signals and nuclear factor-κB (NF-κB) has been reported, and estrogen depletion enhances bone resorption by osteoclasts via NF-κB activation. However, it is unclear when and in which tissues NF-κB is activated after menopause, and how NF-κB acts as a common signaling molecule for postmenopausal weight gain and bone loss. Therefore, we examined the role of NF-κB in bone and energy metabolism following menopause. NF-κB reporter mice, which can be used to measure NF-κB activation in vivo, were ovariectomized (OVX) and the luminescence intensity after OVX increased in the metaphyses of the long bones and perigonadal white adipose tissue, but not in the other tissues. OVX was performed on wild-type (WT) and p65 mutant knock-in (S534A) mice, whose mutation enhances the transcriptional activity of NF-κB. Weight gain with worsening glucose tolerance was significant in S534A mice after OVX compared with those of WT mice. The bone density of the sham group in WT or S534A mice did not change, whereas in the S534A-OVX group it significantly decreased due to the suppression of bone formation and increase in bone marrow adipocytes. Disulfiram, an anti-alcoholic drug, suppressed OVX-induced activation of NF-κB in the metaphyses of long bones and white adipose tissue (WAT), as well as weight gain and bone loss. Overall, the activation of NF-κB in the metaphyses of long bones and WAT after OVX regulates post-OVX weight gain and bone loss.

Role of osteoglycin in the linkage between muscle and bone.

The interaction between muscle tissues and bone metabolism is incompletely understood. We hypothesized that there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We, therefore, performed comparative DNA microarray analysis between mouse myoblastic C2C12 cells transfected with either stable empty vector or ALK2 (R206H), the mutation that constitutively activates the bone morphogenetic protein (BMP) receptor, to search for muscle-derived bone anabolic factors. Twenty-five genes whose expression was decreased to <1/4, were identified; these included osteoglycin (OGN). Stable overexpression of OGN significantly decreased the levels of Runx2 and Osterix mRNA compared with those in cells transfected with vector alone in MC3T3-E1 cells. On the other hand, it significantly enhanced the levels of alkaline phosphatase (ALP), type I collagen (Col1), and osteocalcin (OCN) mRNA as well as ß-catenin and mineralization. A reduction in endogenous OGN level showed the opposite effects to those of OGN overexpression in MC3T3-E1 and mouse calvarial osteoblastic cells. Transient OGN overexpression significantly suppressed the levels of Runx2, Osterix, ALP, Col1, and OCN mRNA induced by BMP-2 in C2C12 cells. The conditioned medium from OGN-overexpressed and OGN-suppressed myoblastic cells enhanced and decreased, respectively, the levels of ALP, Col1, and ß-catenin in MC3T3-E1 cells. Moreover, OGN increased Smad3/4-responsive transcriptional activity as well as Col1 mRNA levels independently of endogenous TGF-ß in these cells. In conclusion, this study suggests that OGN may be a crucial humoral bone anabolic factor that is produced by muscle tissues.

TLE3, transducing-like enhancer of split 3, suppresses osteoblast differentiation of bone marrow stromal cells.

In senile osteoporosis the balance of adipogenesis and osteoblastogenesis in bone marrow stromal cells (BMSCs) is disrupted so that adipogenesis is increased with respect to osteoblastogenesis, and as a result, bone mass is decreased. While the molecular mechanisms controlling the balance between osteoblastogenesis and adipogenesis are of great interest, the exact nature of the signals regulating this process remains to be determined. In general, adipogenesis is a reciprocal relationship with osteoblastogenesis in BMSCs. Recently transducin-like enhancer of split 3 (TLE3), was reported to enhance adipogenesis in pre adipocytes. However, the effect of TLE3 on osteoblast differentiation of BMSCs is completely unknown. Here we report that TLE3 not only enhances adipocyte differentiation in BMSCs but also suppresses osteoblast differentiation. Firstly we examined the expression and localization of TLE3. We found that TLE3 is expressed in the nucleus of bone marrow stromal cells and that over-expression of TLE3 induced adipocyte differentiation and suppressed ALP activity induced by treatment with BMP2 in these cells. In contrast, adipocyte differentiation was decreased and ALP activity increased when endogenous TLE3 was knocked down by shRNA in BMSCs. To examine the mechanism by which TLE3 is able to suppress osteoblast differentiation, we focused on Runx2, a transcription factor essential for osteoblast differentiation. We found that TLE3 strongly suppressed ALP activity and OSE2-luciferase activity induced by Runx2 and this repression of Runx2 by TLE3 occurs via HDACs because treatment with TSA, a class I and II HDAC inhibitor, rescued this repression. In conclusion, we identify TLE3 as a suppressor of BMSC differentiation in osteoblast lineage cells in vitro. Our data suggest that TLE3 activity may be a key in balancing adipocyte and osteoblast differentiation in the adult bone marrow microenvironment.

The unique activity of bone morphogenetic proteins in bone: a critical role of the Smad signaling pathway.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that belong to the transforming growth factor-ß family. BMPs were originally identified based on their unique activity, inducing heterotopic bone formation in skeletal muscle. This unique BMP activity is transduced by specific type I and type II transmembrane kinase receptors. Among the downstream pathways activated by these receptors, the Smad1/5/8 transcription factors appear to play critical roles in BMP activity. Smad1/5/8 transcription factors are phosphorylated at the C-terminal SVS motif by BMP type I receptors and then induce the transcription of early BMP-responsive genes by binding to conserved sequences in their enhancer regions. The linker regions of Smad1/5/8 contain multiple kinase phosphorylation sites, and phosphorylation and dephosphorylation of these sites regulate the transcriptional activity of Smad proteins. Gain-of-function mutations in one BMP type I receptor have been identified in patients with fibrodysplasia ossificans progressiva, a rare genetic disorder that is characterized by progressive heterotopic bone formation in the skeletal muscle. The mutant receptors activate the Smad signaling pathway even in the absence of BMPs, therefore novel inhibitors for the BMP receptor - Smad axis are being developed to prevent heterotopic bone formation in fibrodysplasia ossificans progressiva. Taken together, the data in the literature show that the BMP type I receptor - Smad signaling axis is the critical pathway for the unique activity of BMPs and is a potential therapeutic target for pathological conditions caused by inappropriate BMP activity.

Identification of a novel bone morphogenetic protein (BMP)-inducible transcript, BMP-inducible transcript-1, by utilizing the conserved BMP-responsive elements in the Id genes.

Bone morphogenetic proteins (BMPs) inhibit myogenesis and induce osteoblastic differentiation in myoblasts. They also induce the transcription of several common genes, such as Id1, Id2 and Id3, in various cell types. We have reported that a GC-rich element in the Id1 gene functions as a BMP-responsive element (BRE) that is regulated by Smads. In this study, we analyzed and identified BREs in the 5'-flanking regions of the mouse Id2 and Id3 genes. The core GGCGCC sequence was conserved among the BREs in the Id1, Id2 and Id3 genes and was essential for the response to BMP signaling via Smads. We found a novel BRE on mouse chromosome 13 at position 47,723,740-47,723,768 by searching for conserved sequences containing the Id1 BRE. This potential BRE was found in the 5'-flanking region of a novel gene that produces a non-coding transcript, termed BMP-inducible transcript-1 (BIT-1), and this element regulated the expression of this gene in response to BMP signaling. We found that BIT-1 is expressed in BMP target tissues such as the testis, brain, kidney and cartilage. These findings suggest that the transcriptional induction of the Ids, BIT-1 and additional novel genes containing the conserved BRE sequence may play an important role in the regulation of the differentiation and/or function of target cells in response to BMPs.

Clinically applicable antianginal agents suppress osteoblastic transformation of myogenic cells and heterotopic ossifications in mice.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by progressive heterotopic ossification. FOP is caused by a gain-of-function mutation in ACVR1 encoding the bone morphogenetic protein type II receptor, ACVR1/ALK2. The mutant receptor causes upregulation of a transcriptional factor, Id1. No therapy is available to prevent the progressive heterotopic ossification in FOP. In an effort to search for clinically applicable drugs for FOP, we screened 1,040 FDA-approved drugs for suppression of the Id1 promoter activated by the mutant ACVR1/ALK2 in C2C12 cells. We found that that two antianginal agents, fendiline hydrochloride and perhexiline maleate, suppressed the Id1 promoter in a dose-dependent manner. The drugs also suppressed the expression of native Id1 mRNA and alkaline phosphatase in a dose-dependent manner. Perhexiline but not fendiline downregulated phosphorylation of Smad 1/5/8 driven by bone morphogenetic protein (BMP)-2. We implanted crude BMPs in muscles of ddY mice and fed them fendiline or perhexiline for 30 days. Mice taking perhexiline showed a 38.0 % reduction in the volume of heterotopic ossification compared to controls, whereas mice taking fendiline showed a slight reduction of heterotopic ossification. Fendiline, perhexiline, and their possible derivatives are potentially applicable to clinical practice to prevent devastating heterotopic ossification in FOP.

Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.