An experimental therapy to improve skeletal growth and prevent bone loss in a mouse model overexpressing IL-6.

UNLABELLED: Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory disease. Presently, no treatment regimens are available for these defects in juvenile diseases. We identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in a mouse model overexpressing IL-6. INTRODUCTION: Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory diseases and have a significant impact on patients' quality of life. Presently, no treatment regimens are available for these defects in juvenile diseases. To test a new therapeutic approach, we used growing mice overexpressing the pro-inflammatory cytokine IL-6 (TG), which show a generalized bone loss and stunted growth. METHODS: Since TG mice present increased bone resorption and impaired bone formation, we tested a combined therapy with the antiresorptive modified osteoprotegerin, Fc-OPG, and the anabolic PTH. We injected TG mice with Fc-OPG once at the 4th day of life and with hPTH(1-34) everyday from the 16th to the 30th day of age. RESULTS: A complete prevention of growth and bone defects was observed in treated mice due to normalization of osteoclast and osteoblast parameters. Re-establishment of normal bone turnover was confirmed by RT-PCR analysis and by in vitro experiments that revealed the full rescue of osteoclast and osteoblast functions. The phenotypic recovery of TG mice was due to the sequential treatment, because TG mice treated with Fc-OPG or hPTH alone showed an increase of body weight, tibia length, and bone volume to intermediate levels between those observed in vehicle-treated WT and TG mice. CONCLUSIONS: Our results identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in IL-6 overexpressing mice, thus providing the proof of principle for a therapeutic approach to correct these defects in juvenile inflammatory diseases.

[Residential mobility in old age. Age-appropriate housing as a primary preventive strategy]. / Wohnmobilität im Alter. Altersgerechtes Wohnen als primärpräventive Strategie.

BACKGROUND: Unfavorable living conditions in old age have negative physical, psychological and social implications and increase the risk of institutionalization. The aim of this study is to examine older adults' readiness to relocate to age-appropriate housing versus the wish to stay where they are. METHODS: A total of 103 older men and women were questioned prospectively, face-to-face, regarding their readiness to relocate. In addition to assessing several established predictors of residential mobility, data on objective living situations were collected. The data were subjected to bi- and multivariate analysis. RESULTS: At 70.9%, the proportion of respondents who are not willing to move (stayers) far exceeds the proportion of those who are (movers, 29.1%). Older respondents are more satisfied with their living situation and less likely to move. This study provides empirical evidence for the "satisfaction paradox" with respect to living conditions and for the importance of the subjective standard of living for quality of life in old age. CONCLUSION: Modern housing counseling should more strongly reflect the variety of needs, requirements and living situations of older people today than it has done in the past. It is therefore recommended that the currently dominating paradigm of "aging in place" be critically re-evaluated.

Activity tracker-based intervention to increase physical activity in patients with type 2 diabetes and healthy individuals: study protocol for a randomized controlled trial.

BACKGROUND: One relevant strategy to prevent the onset and progression of type 2 diabetes mellitus (T2DM) focuses on increasing physical activity. The use of activity trackers by patients could enable objective measurement of their regular physical activity in daily life and promote physical activity through the use of a tracker-based intervention. This trial aims to answer three research questions: (1) Is the use of activity trackers suitable for longitudinal assessment of physical activity in everyday life? (2) Does the use of a tracker-based intervention lead to sustainable improvements in the physical activity of healthy individuals and in people with T2DM? (3) Does the accompanying digital motivational intervention lead to sustainable improvements in physical activity for participants using the tracker-based device? METHODS: The planned study is a randomized controlled trial focused on 1642 participants with and without T2DM for 9 months with regard to their physical activity behavior. Subjects allocated to an intervention group will wear an activity tracker. Half of the subjects in the intervention group will also receive an additional digital motivational intervention. Subjects allocated to the control group will not receive any intervention. The primary outcome is the amount of moderate and vigorous physical activity in minutes and the number of steps per week measured continuously with the activity tracker and assessed by questionnaires at four time points. Secondary endpoints are medical parameters measured at the same four time points. The collected data will be analyzed using inferential statistics and explorative data-mining techniques. DISCUSSION: The trial uses an interdisciplinary approach with a team including sports psychologists, sports scientists, health scientists, health care professionals, physicians, and computer scientists. It also involves the processing and analysis of large amounts of data collected with activity trackers. These factors represent particular strengths as well as challenges in the study. TRIAL REGISTRATION: The trial is registered at the World Health Organization International Clinical Trials Registry Platform via the German Clinical Studies Trial Register (DRKS), DRKS00027064 . Registered on 11 November 2021.

Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures.

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption.

The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

Low complication rates in Minimally Invasive Plate Osteosynthesis (MIPO) for proximal humeral fractures at 5 years of follow-up.

INTRODUCTION: Purpose of this study was to analyse the medium term follow-up of minimally invasive plate osteosynthesis (MIPO) for proximal humeral fractures in terms of postoperative shoulder function, radiological outcome and complications. METHODS: 76 consecutive patients with unstable proximal humeral fractures were treated using locking plate with a minimally invasive antero-lateral approach in two surgical centers. Constant score and radiographic evaluation of 74 patients were available at mean follow up of 5 years (minimum 4 years). RESULTS: Mean Constant score was 74 (range to 28-100). Results were comparable in the two centers. Younger patients registered significantly higher scores (p < 0.05). 20 patients (27%) developed complications. Subacromial impingement occurred in 16,2% of cases for varus malreduction (6,7%) and for too proximal plate positioning (9,5%). Primary screws perforation (2,7%), secondary perforation due to cut-out (1,4%), avascular necrosis (AVN) of humeral head (1,4%), partial resorption of greater tuberosity (2,7%), secondary displacement of the greater tuberosity (2,7%) and stiffness (2,7%) were observed. DISCUSSION AND CONCLUSIONS: Even at a medium term follow-up, MIPO for proximal humeral fractures ensured good and reproducible results for most common pattern of fractures. Major complications were lower respect to open procedures, because of soft tissue, deltoid muscle and circumflex vessels sparing.

Extracellular protons acidify osteoclasts, reduce cytosolic calcium, and promote expression of cell-matrix attachment structures.

Because metabolic acids stimulate bone resorption in vitro and in vivo, we focused on the cellular events produced by acidosis that might be associated with stimulation of bone remodeling. To this end, we exposed isolated chicken osteoclasts to a metabolic (butyric) acid and observed a fall in both intracellular pH and cytosolic calcium [( Ca2+]i). These phenomena were recapitulated when bone resorptive cells, alkalinized by HCO3 loading, were transferred to a bicarbonate-free environment. The acid-induced decline in osteoclast [Ca2+]i was blocked by either NaCN or Na3VO4, in a Na+-independent fashion, despite the failure of each inhibitor to alter stimulated intracellular acidification. Moreover, K+-induced membrane depolarization also reduced cytosolic calcium in a manner additive to the effect of protons. These findings suggest that osteoclasts adherent to bone lack functional voltage-operated Ca2+ channels, and they reduced [Ca2+]i in response to protons via a membrane residing Ca-ATPase. Most importantly, acidosis enhances formation of podosomes, the contact areas of the osteoclast clear zone, indicating increased adhesion to substrate, an early step in bone resorption. Thus, extracellular acidification of osteoclasts leads to decrements in intracellular pH and calcium, and appears to promote cell-matrix attachment.

Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts.

Osteoclasts resorb bone by first attaching to the bone surface and then secreting protons into an isolated extracellular compartment formed at the cell-bone attachment site. This secretion of protons (local acidification) is required to solubilize bone hydroxyapatite crystals and for activity of bone collagen-degrading acid proteases. However, the large quantity of protons required, 2 mol/mol of calcium, would result in an equal accumulation of cytosolic base equivalents. This alkaline load must be corrected to maintain cytosolic pH within physiologic limits. In this study, we have measured cytoplasmic pH with pH-sensitive fluorescent compounds, while varying the extracellular ionic composition of the medium, to determine the nature of the compensatory mechanism used by osteoclasts during bone resorption. Our data show that osteoclasts possess a chloride/bicarbonate exchanger that enables them to maintain normal intracellular pH in the face of a significant proton efflux. This conclusion follows from the demonstration of a dramatic cytoplasmic acidification when osteoclasts that have been incubated in bicarbonate-containing medium are transferred into bicarbonate-free medium. This acidification is absolutely dependent on and proportional to medium [Cl-]. Furthermore, acidification is inhibited by the classic inhibitor of red cell anion exchange, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and by diphenylamine-2-carboxylate, an inhibitor of chloride specific channels. However, the acidification process is neither energy nor sodium dependent. The physiologic importance of chloride/bicarbonate exchange is demonstrated by the chloride dependence of recovery from an endogenous or exogenous alkaline load in osteoclasts. We conclude that chloride/bicarbonate exchange is in large part responsible for cytoplasmic pH homeostasis of active osteoclasts, showing that these cells are similar to renal tubular epithelial cells in their regulation of intracellular pH.

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment.

BACKGROUND: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). METHODS: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). RESULTS: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. CONCLUSIONS: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Tissue inhibitor of metalloproteinase-2 protection of matrix metalloproteinase-2 from degradation by plasmin is reversed by divalent cation chelator EDTA and the bisphosphonate alendronate.

The degradation of tissue inhibitor of metalloproteinase (TIMP)-free matrix metalloproteinase (MMP)-2 to proteolytically inactive fragments by plasmin was inhibited in equimolar mixtures of purified TIMP-2 and TIMP-free MMP-2 and was not observed in purified MMP-2-TIMP-2 complexes. Divalent cation chelators EDTA and sodium Alendronate did not inhibit plasmin degradation of TIMP-free MMP-2 but reversed the ability of TIMP-2 to protect MMP-2 from degradation by plasmin. Our data confirm a role for plasmin in the clearance of TIMP-free MMP-2, identify a pivotal role for TIMP-2 in regulating MMP-2 longevity in plasmin-containing environments, and highlight a novel therapeutic use for chelators of divalent cations, including the bisphosphonate Alendronate, in the reversal of TIMP-2 protection of MMP-2 from degradation by plasmin. We propose that these observations are relevant to pathologies that are dependent upon plasmin and MMP-2 activity (e.g., tumor invasion and metastasis).

The distribution of podosomes in osteoclasts cultured on bone laminae: effect of retinol.

Osteoclasts, isolated and purified from the medullary bone of calcium-deficient egg-laying hens, adhere to glass coverslips in vitro by means of specialized protrusions of the ventral membrane, denoted podosomes. These structures represent dotlike close-contact adhesion sites in which most cytoskeletal proteins involved in linking the plasma membrane to microfilaments are organized according to a specific and previously described pattern also shared by many oncogene-transformed cells. We show now that podosomes are not only a feature of osteoclasts adhering to artificial glass surfaces but are also present in the ventral membrane of osteoclasts adhering to bone laminae. Moreover, the quantity and the topography of podosomes may be modulated by retinol, which increases bone-resorbing activity of osteoclasts both in vivo and in vitro. A comparative transmission electron microscopy study of osteoclasts adhering on bone laminae in vitro or in vivo indicates that podosomes with identical features are present in the clear zone of the osteoclasts in either condition. Since podosomes are the sealing structures of the clear zone, podosome formation may represent one of the modifications involved in the reorganization process of the osteoclast that precedes bone resorption.

Effect of vitamin A on bone resorption: evidence for direct stimulation of isolated chicken osteoclasts by retinol and retinoic acid.

The effects of retinol (vitamin A) and retinoic acid on primary cultures of isolated chicken osteoclasts have been studied. The experiments were performed to establish the direct actions of these two agents on the organization of cytoskeletal structures, on the acid phosphatase contents, and on the bone resorption activities of these cells. The results showed that by treating the cultures with retinol or retinoic acid, from 10(-8) to 10(-5) M, there were dose-related responses of the osteoclasts. Adhesion to the substratum was stimulated by increasing the number of cells exhibiting the specialized dot-like adhesion structures, or podosomes, which represent the active part of the sealing zone. The treatments also induced rearrangement of the microtubular patterns with reversible depolymerization of microtubules. Acid phosphatase activity was significantly higher both in vitamin A-treated osteoclasts and in their media. When [3H]proline-labeled bone particles were added to the retinoid-treated osteoclasts, the release of [3H]proline was increased significantly compared to controls. These results suggest that the two vitamin A metabolites cause several modifications of the metabolic status of isolated osteoclasts that result in augmented rates of bone resorption.

Apparent cure of a newborn with malignant osteopetrosis using prednisone therapy.

A newborn girl with hemorrhagic purpura, suspected neonatal sepsis, and pale and dry skin was lethargic with remarkable hepatosplenomegaly, convergent strabismus, severe anemia, and elevated alkaline phosphatase activity. Radiographs showed a generalized increase in bone density, small medullary cavities, sclerosis of the skull and vertebrae, transverse wavy stripes of sclerotic bone in the metaphyses, and bone-in-bone appearance in phalanges of hands and feet. On this basis, she was diagnosed with malignant infantile osteopetrosis. On the first day of life, the infant was given a blood transfusion and vitamin K (1 mg intravenously [iv]). Corticosteroid therapy was started with prednisone (2 mg/kg per day). She showed marked improvement of symptoms. On the 26th day and 42nd day of life, she received additional blood transfusions. On the 49th day, the patient was discharged and corticosteroid therapy was continued at a regimen of 5 mg/day. Subsequent blood sample analyses revealed normal values for age. At 1 year of life, a bone marrow sample showed normal white and red cell lineages. X-ray confirmed attenuation of the bone sclerosis; therefore, bone marrow transplantation (BMT) was not implemented. At the age of 1.5 years, prednisone therapy was discontinued gradually and withdrawn before the age of 2 years. Subsequent follow-up showed normalization of all radiological and hematologic parameters. At present, the patient is 3 years old and appears healthy with apparently complete regression of the disease.

New model for bone resorption study in vitro: human osteoclast-like cells from giant cell tumors of bone.

Cells harvested from 12 human giant cell tumors of bone and kept in culture for several passages were characterized for bone-resorbing capability, total and tartrate-resistant acid phosphatase activity, response to the calciotropic hormone calcitonin, cell proliferation, multinucleation after passages, and presence of calcium sensing. Cells obtained from three tumors presented a complete panel of osteoclast characteristics and maintained their multinuclearity after several passages. Cells from four other tumors increased their cAMP levels after treatment with calcitonin, and the other five apparently consisted of cells of stromal origin. These human cell populations with osteoclast characteristics may provide valid in vitro models for the investigation of osteoclastic differentiation and activity.

Colony stimulating factor-1-induced osteoclast spreading depends on substrate and requires the vitronectin receptor and the c-src proto-oncogene.

The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1-induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1-induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the alpha vbeta3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1-induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1-induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata.

Oligodeoxynucleotide targeted to the alphav gene inhibits alphav integrin synthesis, impairs osteoclast function, and activates intracellular signals to apoptosis.

The alphav integrin subunit is highly expressed in osteoclasts where it dimerizes with beta1 and beta3 subunits to form receptors for vitronectin and bone sialoproteins. Inhibition of osteoclast adhesion and function has previously been achieved by alphavbeta3 antibodies or Arg-Gly-Asp-containing peptides which have the disadvantages of blocking a single receptor type, or of being rather nonspecific, respectively. Here we show that alphav integrin expression in rabbit osteoclasts can be inhibited by partially phosphorothioated antisense oligodeoxynucleotide (ODN) spanning the adenine-uracil-guanine (AUG) translational start site of the human/rabbit alphav gene, a procedure which offers the advantage of affecting all the alphav receptors with high efficiency. The alphav antisense ODN caused a dose-dependent, substrate-specific reduction of osteoclast adhesion and bone resorption. Control ODNs, such as sense, inverted, and mismatch, were without effect, providing evidence of specificity of the antisense reagent. It is likely as a consequence of loss of substrate interaction, the antisense ODN induced osteoclast retraction and apoptosis, increase of the cyclin/cyclin-dependent kinase complex inhibitor p21WAF1/CIP1, and inhibition of the cell survival gene, bcl-2. Although the expression of the cell death-promoting gene, bax, remained unchanged, a reduction of the bcl-2/bax ratio, known to underlie the intracellular signal to apoptosis, was observed. This finding led us to hypothesize that these changes could provide a link between reduction of alphav synthesis and osteoclast programmed death. In conclusion, this study provides novel insights into the use of alphav antisense ODN as an efficacious mechanism for blocking osteoclast function and underscores for the first time the involvement of integrins in bone cell apoptosis. In vivo studies may verify potential application of this ODN as alternative therapy for bone diseases.

Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclast-specific adhesion structures.

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

Tartronates: a new generation of drugs affecting bone metabolism.

In the search for a new class of bone-sparing agents for treating osteopenic disorders, we hypothesized that tartronic acid derivatives, sharing the chemical characteristics both of bisphosphonates and of Gla residues contained in matrix proteins such as osteocalcin, could positively affect bone metabolism. A series of tartronates was therefore tested for their ability to affect bone metabolism. In vitro resorption tests were performed examining pit formation by freshly isolated rat and rabbit osteoclasts plated onto bone slices and exposed to the drugs for 48 h. Tartronates bearing a linear side-chain (DF 1222 and DF 1363A) were the most effective in inhibiting pit excavation in the pM-nM range. Tartronates did not affect osteoclast viability, number, adhesion, or tartrate resistant acid phosphatase activity. Transient cell retraction was observed in osteoclasts plated onto glass and exposed to DF 1222. The maximal effect was seen in cells treated for 4 h at a concentration of 1 pM. DF 1222 accelerated mineralization in cultures of periosteal cells without affecting other osteoblast-like functions. This product was therefore tested in vivo in ovariectomized mice. Bone mass in femur was evaluated, by ash gravimetry, 21 days after ovariectomy. Unfortunately, DF 1222, the most active of tartronates in vitro, was inactive in this test because of its high hydrophilicity and the subsequent too short residence time. On the contrary, its tetrahydropyranyl ether derivative, DF 1363A, endowed with a significantly higher lipophilicity, showed a dose-dependent bone-sparing effect when administered subcutaneously at 10, 30, and 100 mg/kg/die, thus confirming the activity seen in in vitro tests. Because of their feasible parallel effect on both bone resorption and formation, tartronate derivatives may be tested to candidate this class of products for clinical studies.

Beta1 integrin antisense oligodeoxynucleotides: utility in controlling osteoclast function.

The involvement of beta1 integrins in osteoclast function has been investigated by utilising an antisense oligodeoxynucleotide (ODN) approach. 18-mer antisense and control phosphorothioate ODNs were made to a conserved internal region of beta1 integrin sequence (nucleotide positions 1634-1651 of the human beta1 fibronectin receptor). These were tested on rabbit osteoclasts for anti-adhesive and resorptive effects mediated by alphaVbeta3 and alpha2beta1, the major integrins of osteoclasts. Antisense, but not control, beta1 ODNs inhibited osteoclast adhesion to collagen-coated glass (by up to 70%), but not to glass coated with vitronectin, fibronectin or fibrinogen. Adhesion to dentine and subsequent resorption were also inhibited (up to 60%) in a sequence-specific manner. The mechanism of action was verified using both a melanoma cell line, DX3, which expresses multiple integrins at high level including alphaVbeta3 and alpha2beta1, and in a rabbit osteoclast marrow culture (BMC) system. Exposure of DX3 cells to antisense ODN for up to 48 hours reduced adhesion to FCS- and collagen-coated glass, and concomitantly inhibited beta1 protein expression assessed by FACS and Western blot analysis; expression of other integrin subunits, alphaV and beta3, was unaffected. Similarly, the beta1 protein levels in the BMC were reduced by > 75% without any effect on actin expression. These data reveal the utility of antisense ODNs in exploring osteoclast biology and further define the functional role of osteoclastic beta1 integrin(s).