In Vitro andIn Vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene

It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation bothin vivo andin vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effectin vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formationin vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both nontransduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potentialin vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation bothin vitro andin vivo. Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357–360, 1998). SLC, JL, and NMW have contributed equally to this work and therefore should be considered first authors.     

Osteogenic Protein-1 Stimulates mRNA Levels of BMP-6 and Decreases mRNA Levels of BMP-2 and -4 in Human Osteosarcoma Cells

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively. BMP-2 mRNA expression, as examined by Northern blot analysis, was below detectable limits in SaOS-2 cultures. These results demonstrate that OP-1 modulates the mRNA expression of related members of the BMP family, suggesting a possible mode of action of OP-1 on the growth and differentiation of cells in the osteoblastic lineage in vitro. Received: 7 May 1996 / Accepted: 24 September 1996     

Expression of BMP-2 by Rat Bone Marrow Stromal Cells in Culture

To investigate the role of bone morphogenetic protein (BMP-2) in ossifying rat bone marrow stromal cell cultures, we determined the population of fibroblast-like stromal cells that expressed BMP-2 immunocytochemically (anti-rhBMP-2 monoclonal antibody), and compared that to alkaline phosphatase (AP) and collagen synthesis formed in culture over a 4-week period in control and dexamethasone-supplemented mineralizing media. In control media, the percentage of BMP-2-positive stromal cells (BMP-2⁺) increased from 12 to 25% within the first 4 days of culture. In mineralizing media, the level of BMP-2⁺ cells was significantly increased (43–44%). The intensity of immunostaining gradually increased with time. The levels of AP were undetectable at 1 week in both control and mineralizing media, but increased gradually over the next 2 weeks and peaked at 3 weeks. ALP levels were significantly greater in cultures grown in mineralizing medium (P < 0.05 at 3 weeks, P < 0.01 at 4 weeks). Collagen synthesis peaked and was significantly greater at 3 weeks (P < 0.05) in cultures grown in mineralizing medium. The levels of AP and collagen synthesis most closely reflected the changes in the percentage of BMP-2⁺ cells from 7 to 28 days. Though these changes may reflect a primary action of BMP-2 on marrow osteoprogenitor-like stromal cells, they do not exclude a mechanism that involves the induction of other members of the BMP family known to stimulate AP and collagen synthesis. We conclude that BMP-2 expression in cultures of fibroblast-like marrow stromal cells is enhanced when those cells are induced to become osteoblasts by exposure to dexamethasone. Received: 30 October 1997 / Accepted: 24 June 1998     

A Novel Human Bone Marrow Stroma-Derived Cell Line TF274 Is Highly Osteogenic In Vitro and In Vivo

A novel, immortalized, human bone marrow stroma-derived cell line TF274 is described which has the ability to form bone both in vitro and in vivo. Under basal conditions these cells expressed alkaline phosphatase (ALP) and type I collagen genes which are characteristic of the osteoblast phenotype. ALP levels were upregulated in the presence of osteotropic agents such as parathyroid hormone (PTH), transforming growth factor beta (TGF-β), and BMP-2. In addition, PTH also increased cAMP levels in these cells. The capacity of these cells to form bone in vitro was evaluated by culturing them in the presence of L-ascorbic acid and β-glycerophosphate. Matrix mineralization in these cultures was assessed by Alizarin Red staining and increased ⁴⁵Ca uptake. Under these conditions mineralized nodule formation was observed in less than 2 weeks. Northern analysis of TF274 cells at various times during the mineralization process indicated a temporal expression of the osteocalcin gene that is typically associated with differentiating osteoblasts. The osteogenic nature of TF274 cells was confirmed in vivo using the severe combined immunodeficient (SCID) mouse model. Antibodies to human leukocyte antigens (HLA), class I antigens, and human OK^a blood group antigen were used to demonstrate that the lesions formed were of human origin. By 21 days, the lesion consisted of a homogeneous focus of ALP-positive cells containing areas of mineralized bone lined with tartarate-resistant acid phosphatase (TRAP) positive osteoclasts. Thus, the TF274 cells exhibit osteogenic potential both in vitro and in vivo. This immortalized cell line represents a consistent source of cells that can be used to study human osteoblast differentiation both in vitro and in vivo. Received: 30 July 1997 / Accepted: 23 January 1998     

腺病毒骨形态发生蛋白2绿色荧光蛋白基因转染兔骨髓基质干细胞成骨及示踪作用

目的 观察腺病毒介导的人骨形态发生蛋白绿色荧光蛋白基因(Ad-GFP-hBMP-2)转染对骨髓间质干细胞(bMSCs)成骨能力的影响.方法 取日本大耳白兔4只自双侧股骨远端抽取骨髓培养bMSCs.以Ad-GFP-hBMP-2基因(实验组)及Ad-GFP(对照组)基因转染bMSCs后,用ALP检测试剂盒检测两组细胞的ALP活性;原位杂交检测两组细胞I型胶原的表达;Western blot 检测细胞中BMP-2的表达.将转染后24 h的bMSCs接种到裸鼠体内,术后第4、8、12周观察成骨情况.结果 转Ad-GFP-hBMP-2基因组和Ad-GFP组各时间段ALP分泌量差异分别有统计学意义(P<0.01);实验组I型胶原原位杂交实验组为阳性.实验组成骨阳性率为90%,对照组为40%.结论 bMSCs经Ad-GFP-hBMP-2基因转染后能高效表达BMP-2并诱导成骨.腺病毒介导人BMP-2转基因可以提高bMSCs的成骨能力.     

Basic Fibroblast Growth Factor in the Presence of Dexamethasone Stimulates Colony Formation, Expansion, and Osteoblastic Differentiation by Rat Bone Marrow Stromal Cells

Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, β-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect. Received: 17 January 1997 / Accepted: 30 July 1998     

Bone Morphogenetic Protein 2 (BMP-2) Enhances BMP-3, BMP-4, and Bone Cell Differentiation Marker Gene Expression During the Induction of Mineralized Bone Matrix Formation in Culturesof Fetal Rat Calvarial Osteoblasts

Normal bone formation is a prolonged process that is carefully regulated and involves sequential expression of growth regulatory factors by osteoblasts as they proliferate and ultimately differentiate. Since this orderly sequence of gene expression by osteoblasts suggests a cascade effect, and BMP-2 is capable of initiating and maintaining this effect, we examined the effects of BMP-2 on expression of other BMPs and compared these effects with the expression pattern of bone cell differentiation marker genes in primary cultures of fetal rat calvarial (FRC) osteoblasts. To examine the gene expression profile during bone cell differentiation and bone formation, we also examined the effects of rBMP-2 on bone formation in vivo and in vitro. rBMP-2 stimulated bone formation on the periosteal surface of mice when 500 ng/day rBMP-2 was injected subcutaneously. When rBMP-2 was added to primary cultures of FRC osteoblasts, it accelerated mineralized nodule formation in a time and concentration-dependent manner (10–40 ng/ml). rBMP-2 (40 ng/ml) enhanced BMP-3 and -4 mRNA expression during the mineralization phase of primary cultures of FRC osteoblasts. Enhancement of BMP-3 and -4 mRNA expression by rBMP-2 was associated with increased expression of bone cell differentiation marker genes, alkaline phosphatase (ALP), type I collagen, osteocalcin (OC), osteopontin (OP), and bone sialoprotein (BSP). These results suggest that BMP-2 enhances expression of other BMP genes during bone cell differentiation. BMP-2 may act in a paracrine fashion in concert with other BMPs it induces to stimulate bone cell differentiation and bone formation during remodeling. Received: 27 November 1995 / Accepted: 19 July 1996     

Age-Related Changes in the Expression of Cadherin-11, the Mesenchyme Specific Calcium-Dependent Cell Adhesion Molecule

Cadherin-11 is a calcium-dependent cell adhesion molecule that is expressed in cells of the mesenchymal lineage during embryonic development. In this study we show, for the first time, that cadherin-11 gene is expressed in the bone marrow and bone cells obtained from rabbits of various age groups. Furthermore, a quantitative measurement of gene expression revealed that cadherin-11 was expressed in young rabbits (6 week-old: open epiphysis) at a level of 6.7 × 10⁵± 0.7 × 10⁵ molecules; in mature rabbits (8–10 month-old: closed epiphysis) at 11 × 10⁵± 0.9 × 10⁵ molecules; and in aged rabbits (4–5 year-old) at a level of 1.2 × 10⁵± 0.2 × 10⁵ molecules/μg total RNA. The relative level of cadherin-11 gene expression in mature rabbit marrow was found to be approximately 50% greater than in young rabbits. However, aged animals showed a reduction in cadherin-11 specific gene expression of greater than 900% as compared with mature animals. Age-related changes in bone remodeling/turnover lead to reduced bone density and high fracture risk, and since cadherins play a crucial role in tissue morphogenesis, this marked decrease may represent an index of the aging process in bone. Received: 2 January 1997 / Accepted: 29 September 1997     

慢病毒介导BMP-2过表达质粒转染骨髓间充质干细胞联合丝素蛋白支架向成骨细胞转化的实验研究

目的：探究慢病毒介导BMP-2过表达质粒转染骨髓间充质干细胞联合丝素蛋白支架向成骨细胞转化的作用效果。方法：构建慢病毒BMP-2过表达载体,培养骨髓间充质干细胞,构建细胞核支架的联合培养体系,体外实验利用茜素红染色和碱性磷酸酶染色检测骨髓间充质干细胞的成骨转化。选择10只新西兰大白兔,体重3.2～4.5 kg,平均3.9 kg;年龄（2.89±0.45）岁;使用口腔钻在兔子胫骨钻孔（长度5 mm、宽度2 mm、深度3 mm的锥形胫骨缺损）构建兔子胫骨骨缺损模型,HE染色观察动物模型内骨缺损的修复。实验组造模后植入丝素蛋白支架+转染BMP-2过表达载体骨髓间充质干细胞复合物,阴性对照组造模后植入丝素蛋白支架+未转染骨髓间充质干细胞复合物。结果：实验组（丝素蛋白支架+转染BMP-2过表达载体骨髓间充质干细胞复合物）中支架表面黏附的细胞与对照组（丝素蛋白支架+未转染骨髓间充质干细胞）相比,细胞数明显增多。实验组细胞外基质分泌与对照组相比,支架间细胞外基质含量明显增多。对照组支架表面元素EDX分析显示钙离子含量为0.22%,实验组支架表面元素EDX分析显示钙离子含量为0.86%,可见实验组诱导钙离子形成的能力要比对照组强。钙结节茜素红染色结果显示,对照组肉眼观无明显变化,镜下观察可见少量钙结节点。实验组肉眼观可见明显红色区域染色,镜下观察可见大量钙结节点。碱性磷酸酶染色结果显示,对照组肉眼观无明显变化,镜下观察未见明显变化。实验组肉眼观可见紫色区域染色,镜下观察可见ALP染色呈强阳性。丝素蛋白支架与骨髓间充质干细胞联合培养体系可以对软骨缺损有较好的修复作用,转染BMP-2骨髓间充质干细胞后修复作用明显优于未转染组。HE染色结果显示,对照组炎性细胞减少,支架略有消失。实验组炎性细胞明显减少,支架消失,血管生成。结论：慢病毒介导BMP-2过表达质粒可以促进BMSC向骨细胞的分化作用,并且分泌更多的含Ca²⁺成分的细胞外基质,从而发挥其促进骨缺损修复的作用。     

The Immunohistochemical Localization of Fas and Fas Ligand in Jaw Bone and Tooth Germ of Human Fetuses

The cellular localization and roles of bone morphogenetic protein (BMP)-2 and apoptosis-associating factors in human orofacial development remain unclear. In this study, BMP-2, osteocalcin, and TGF-β, which are bone-differentiating markers, apoptosis-associating factors (i.e., Bcl-2, Bax, Fas, and Fas ligand), apoptotic cells detected by the in situ 3′-end labeling method (TUNEL), and proliferating cell nuclear antigen (PCNA) were immunohistochemically examined in the heads (in particular, the jaw bone and tooth germs) of human fetuses of 11-week pregnancy. BMP-2 was positive in osteoblasts and newly formed osteoid of the incisive and palatal bone of the maxilla and the mandible, which indicated that BMP-2 was exclusively involved in intramembranous ossification in the human fetal head. Fas was positive in the cytoplasm of osteocytes and a few osteoblasts. In contrast, Fas ligand was positive in the cytoplasm of osteoblasts and abundant in the stroma of the osteoblastic layer, periosteum, and perichondrium. The Fas ligand in the stroma was recognized as the soluble form, which was possibly produced by osteoblasts. TUNEL-positive apoptotic cells were found in a few osteocytes and a few osteoblastic cells in new bone, and in monocytes of degenerate Meckel's cartilage. The induction of apoptosis observed in monocytes seems to be caused via a Fas-Fas ligand cell death system, because some of these monocytes were Fas-positive, and most of them were Fas ligand-positive. Interestingly, the abundant soluble Fas ligand observed in the periosteum probably protects the bone-formative zone from the invasion of the activated lymphocytes by binding to Fas expressing in these lymphocytes and killing these cells. Fas and Fas ligand were focally positive in the dental lamina and inner enamel epithelium and cusps of the enamel organ, nevertheless, the presence of TUNEL-positive cells was very rare. Bcl-2 was clearly and Bax was weakly positive in the cells throughout the dental lamina and enamel organ. These findings indicated that Fas-mediated apoptosis was inhibited by the Bcl-2 family in the development of teeth. Received: 22 January 1998 / Accepted: 2 December 1999     

Bone formation following transplantation of genetically modified primary bone marrow stromal cells.

Bone marrow stromal cells contain mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues; in the presence of BMP-2, for example, they differentiate into osteoblasts. We constructed replication-deficient adenoviral vectors encoding human BMP-2 (BMP-2/Ad) or BMP-4 (BMP-4/Ad) and used them to transduce primary bone marrow stromal cells from the femurs of four-week-old female C3H mice, which then expressed and processed functional BMP-2 or BMP-4 protein. Enzyme assays and histochemical staining showed both groups of cells to possess alkaline phosphatase activity, a marker of differentiation into osteoblasts, though the activity was higher in cells transduced with BMP-2/Ad. When BMP-2/Ad-transduced cells were injected into the thigh muscles of immunocompetent C3H mice, ossicle development was detected on radiographs within four weeks after injection. Moreover, histological analysis indicated that newly developed ossicles contain mature osseous components, including cortical bone and bone marrow, within eight weeks. Thus, syngeneic transplantation of genetically modified primary bone marrow stromal cells induced bone formation in immunocompetent mice, perhaps indicating its potential for use in the development of therapeutic protocols aimed at enhancing bone formation.     

Phenotypic and Molecular Heterogeneity in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia (myositis) ossificans progressiva (FOP) is an extremely rare inherited disorder in which progressive ossification of major striated muscles, often following injury, is associated with abnormal skeletal patterning. Altered expression of bone morphogenetic proteins may be a contributory cause. To examine this hypothesis, we compared the patterns of expression of bone morphogenetic proteins (BMPs) mRNAs from lymphoblastoid cell lines from two small multigenerational families with autosomal dominant transmission of FOP. Although affected members of both families showed the characteristic phenotype of FOP, one family was more severely affected than the other. Expression of mRNAs for BMP-1, 2, 3, 5, and 6 mRNAs were not detected within the more severely affected family, but BMP-4 mRNA was expressed in affected but not unaffected members of this family. The results of linkage exclusion analysis using a highly polymorphic microsatellite marker near the BMP-4 gene were consistent with linkage of FOP and BMP-4 in this family. Within the less severely affected family, affected and unaffected members showed similar levels of mRNA expression of BMPs 1, 2, 4, and 5, and linkage of FOP to the BMP-4 gene was excluded. It is concluded that clinical, radiographic, and biochemical data in these two families with FOP establish clinical and molecular heterogeneity and also suggest the possibility of genetic heterogeneity. Received: 22 January 1998 / Accepted: 9 December 1998     

Bone Morphogenetic Protein-2 Stimulates Differentiation of Cultured Spinal Ligament Cells from Patients with Ossification of the Posterior Longitudinal Ligament

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by heterotopic bone formation occurring in spinal ligament, causing severe compression myelopathy. In order to investigate the mechanism of OPLL development, we isolated spinal ligament cells from OPLL patients as well as non-OPLL patients, and established 10 OPLL cell lines and 7 non-OPLL cell lines, respectively. We analyzed the effects of bone morphogenetic protein-2 (BMP-2) on these cells with respect to alkaline phosphatase (AP) activity, DNA synthesis, and collagen production. BMP-2 caused a significant increase of AP activity in 4 OPLL cell lines, whereas the activity did not change in any non-OPLL cells. Among OPLL cells, BMP-2 stimulated DNA synthesis in four cell lines and procollagen type I carboxyl-terminal peptide (PICP) synthesis in five cell lines. Some non-OPLL cells also responded to BMP-2, as there was an increase of DNA synthesis in three cell lines and PICP synthesis in one cell line. These data collectively indicate that BMP-2 preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. OPLL cells, therefore, exhibit a different response to BMP-2 than non-OPLL cells, suggesting that the expression of BMP receptor(s) and/or the signal transduction initiated by BMP-2 in the spinal ligament cells of OPLL patients somewhat deviate from those in normal spinal ligament cells. Such abnormal characteristics of OPLL cells as described here provide some clues to the clarification of the pathogenesis of OPLL. Received: 1 April 1996 / Accepted: 19 July 1996     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

Soluble Factors Secreted by Macrophage-like Cells In Vitro Cause Osteoprogenitor Cell Detachment

Explant cultures of adult rabbit bone were obtained from a variety of skeletal sites, with the intention of using the cells in a new type of bone graft. The explant cultures contained large numbers of esterase-positive, alkaline phosphatase-negative, TRAP-negative macrophage-like cells (MP), as well as osteoblast-like osteoprogenitor (OP) cells (alkaline phosphatase-positive). OP numbers were assessed by cell counts and MTT assay. The presence of the MP cells appeared to give rise to a reduction in OP numbers in culture, through cell detachment and cell lysis. In addition, after passaging, many OP were unable to reattach to the culture vessels in the presence of MP, depending upon the surface area available for reattachment. The presence of tumor necrosis factor α (TNFα) in culture medium from these cell cultures was demonstrated by a specific enzyme-linked immunosorbent assay (ELISA). A direct relationship was demonstrated between MP numbers and TNFα concentration and an inverse relationship between MP numbers and OP numbers in co-cultures. This was also found when OP cells were exposed to different concentrations of rTNFα, in place of the MP. Incubation with anti-TNFα inhibited the effect of MP and TNFα on OP. These results suggest that MP are able to cause detachment and lysis of OP cells, probably by secretion of a soluble factor in vitro which may be TNFα. Received: 3 January 1997 / Accepted: 5 March 1998     

Multiple Cellular Phenotypes in an Insertional Mutation in Mouse Affecting Bone Development

The 6093 line of transgenic mice exhibits altered bone development as a result of an insertional mutation by the transgene. Female transgenic mice show a marked kyphosis as early as 2 weeks of age. Vertebrae from female mice have lower total bone area and mineral content than age-matched, gender-matched controls, although the bone mineral density is not changed. The femur and tibia exhibit the opposite effect—increased bone area and mineral content. Fluorescent bone label experiments indicated an increased rate of bone mineral deposition in the femur during the early postnatal growth period, and bone marrow from femurs of 6093 females had increased numbers of fibroblast colony-forming units. Transgenic females also are obese and have altered thymocyte development, suggesting that the insertional mutation affects multiple cell populations. We hypothesize that these phenotypes arise as a result of an alteration in the function or developmental potential of a stromal cell or mesenchymal stem cell. Received: 17 April 2000 / Accepted: 24 August 2000 / Online publication: 22 December 2000     

Endothelin-1 and Paget's Bone Disease: Is There a Link?

The abundance of endothelial cells in bone marrow and the proximity of these cells to osteoclasts and osteoblasts suggest a role for endothelin-1 (ET-1) on bone metabolism. In vitro, the direct contact with bone endothelial cells induces osteoclastic progenitors to differentiate into mature elements. Recently it has been reported that ET-1 inhibits osteoclastic bone resorption and cell mobility through a specific receptor on osteoclasts; other authors demonstrated that ET-1 exerts a mitogenic activity on osteoblast-like cells (MC3T3) by stimulating tyrosin phosphorylation. We measured ET-1 circulating levels in patients with active Paget's bone disease, a condition with accelerated bone turnover. For the study we recruited 11 patients with Paget's bone disease (5F, 6M; mean age 68.2 ± 3.6) in the acute stage of the disease; 10 healthy subjects (7F, 3M; mean age 66.5 ± 3.9) were also enrolled as controls. Plasma ET-1 levels were measured with RIA kits provided by Nichols Institute. Patients showed significantly (P < 0.01) higher ET-1 circulating levels than controls (6.35 ± 1.9 versus 3.4 ± 1.2 pg/ml) with a positive correlation (r = 0.63; P= 0.038) with serum alkaline phosphatase (ALP), but not with urinary hydroxyproline. The higher levels of ET-1 in our patients suggest a physiopathological role for this peptide in the disease and, could perhaps represent a new useful marker of Paget's bone disease activity. Received: 29 April 1997 / Accepted: 20 February 1998     

Fluoride Treatment Increased Serum IGF-1, Bone Turnover,and Bone Mass,but Not Bone Strength,in Rabbits

We hypothesized that fluoride partly acts by changing the levels of circulating calcium-regulating hormones and skeletal growth factors. The effects of oral fluoride on 24 female, Dutch-Belted, young adult rabbits were studied. The rabbits were divided into two study groups, one control and the other receiving about 16 mg fluoride/rabbit/day in their drinking water. After 6 months of fluoride dosing, all rabbits were euthanized and bone and blood samples were taken for analyses. Fluoride treatment increased serum and bone fluoride levels by over an order of magnitude (P < 0.001), but did not affect body weight or the following serum biochemical variables: urea, creatinine, phosphorus, total protein, albumin, bilirubin, SGOT, or total alkaline phosphatase. No skeletal fluorosis or osteomalacia was observed histologically, nor did fluoride affect serum PTH or Vitamin D metabolites (P > 0.4). BAP was increased 37% (P < 0.05) by fluoride; serum TRAP was increased 42% (P < 0.05); serum IGF-1 was increased 40% (P < 0.05). Fluoride increased the vertebral BV/TV by 35% (P < 0.05) and tibial ash weight by 10% (P < 0.05). However, the increases in bone mass and bone formation were not reflected in improved bone strength. Fluoride decreased bone strength by about 19% in the L5 vertebra (P < 0.01) and 25% in the femoral neck (P < 0.05). X-ray diffraction showed altered mineral crystal thickness in fluoride-treated bones (P < 0.001), and there was a negative association between crystal width and fracture stress of the femur (P < 0.02). In conclusion, fluoride's effects on bone mass and bone turnover were not mediated by PTH. IGF-1 was increased by fluoride and was associated with increased bone turnover, but was not correlated with bone formation markers. High-dose fluoride treatment did not improve, but decreased, bone strength in rabbits, even in the absence of impaired mineralization. Received: 5 November 1996 / Accepted: 3 January 1997