H. Fleisch

Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl 2 MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats.

Young male rats (70–90 g) were treated for various periods with several doses of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) or disodium dichloromethylene diphosphonate (Cl2MDP). Effects of treatment on the changes in the thickness, growth and mineralization of proximal growth plate and metaphysis of the tibia were assessed histologically and by micro-radiography. High doses (10 or 30 mg P/kg/day) of EHDP impaired mineralization of the growth cartilage, which became increased in thickness, and of the osteoid in the metaphysis and diaphysis. Matrix formation continued, although at a diminished rate. High doses (10 or 30 mg P/kg/day) of Cl2MDP produced a different effect. There was no inhibition of mineralization, but there was a marked impairment of normal metaphyseal remodelling, with persistence of columns of calcified cartilage. Resorption at the periosteal surface in the metaphysis was also inhibited, so that the metaphysis became club-shaped. Osteoclasts were present in large numbers in the metaphysis, but their appearance was abnormal and similar to that seen in human osteopetrosis.RésuméDes jeunes rats mâles (70–90 g) ont été traités pendant des laps de temps variables avec différentes doses d’éthane-1-hydroxy-1,1-diphosphonate disodique (EHDP) ou de dichlorométhylène diphosphonate disodique (Cl2MDP). Les effects de ces traitements sur l’épaisseur, la croissance et la minéralisation du cartilage de conjugaison et de la métaphyse du tibia ont été étudiés par des méthodes histologiques et microradiographiques. Des doses élevées (10 ou 30 mg P/kg/jour) d’EHDP empêchent la minéralisation de l’ostéoide dans la métaphyse et la diaphyse et celle du cartilage épiphysaire dont l’épaisseur augmente. La formation de la matrice osseuse se poursuit, mais à une vitesse réduite. Le Cl2MDP à doses élevées (10 ou 30 mg P/kg/jour) produit un effet différent. On n’observe aucune inhibition de la minéralisation, mais une diminution nette du remodàlement normal de la métaphyse, où des colonnes de cartilage calcifié persistent. Il y a également, pour la métaphyse, une inhibition de la résorption à la surface périostale provoquant une métaphyse en forme de massue. Dans la métaphyse on note la présence de nombreux ostéoclastes d’apparence anormale qui ressemblent à ceux observés dans l’ostéopétrose humaine.ZusammenfassungJunge männliche Ratten (70–90 g) wurden während verschiedenen Zeitabschnitten mit unterschiedlichen Dosen von Dinatrium Äthan-1-hydroxy-1,1-Diphosphonat (EHDP) oder Dinatrium Dichloromethylen-Diphosphonat (Cl2MDP) behandelt. Die Wirkung der Behandlung auf die Veränderungen in Dicke, Wachstum und Mineralisation der proximalen Wachstumsplatte und Metaphysis der Tibia wurde histologisch und mikroradiographisch untersucht. Hohe Dosen (10 oder 30 mg P/kg/Tag) von EHDP beeinträchtigten die Mineralisation des Wachstumsknorpels, welcher breiter wurde, und des Osteoids in der Metaphyse und Diaphyse. Die Matrixbildung ging weiter, jedoch weniger schnell. Hohe Dosen (10 oder 30 mg P/kg/Tag) von Cl2MDP hatten eine andere Wirkung. Die Mineralisation wurde nicht gehemmt, aber der normale Metaphysenumbau wurde merklich gestört, wobei Säulen von verkalktem Knorpel bestehen blieben. Die Resorption auf der Periostoberfläche in der Metaphyse wurde ebenfalls gehemmt, so daß die Metaphyse keulenförmig wurde. Osteoclasten traten in großen Mengen in der Metaphyse auf, aber ihr Aussehen war abnormal und glich demjenigen, das bei der menschlichen Osteopetrose beobachtet wird.

The binding of pyrophosphate and two diphosphonates by hydroxyapatite crystals

The binding on hydroxyapatite has been studied of inorganic pyrophosphate (PPj) and two diphosphonates, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethylene diphosphonate (Cl2MDP). The binding is greatest for PPi, less for EHDP and least for Cl2MDP. The binding of all three is accompanied by a release of orthophosphate into the solution and an uptake of calcium by the crystals. A competition exists between EHDP and PPi, EHDP being more potent in displacing PPi than the reverse. An analysis with Scatchard plots suggests that the binding is not restricted to one class of binding sites with equal affinity and that the affinity of the first site is in the order EHDP>PPi>Cl2MDP. The results correlate well with previous data on the effect of these compounds on apatite crystal formation and dissolutionin vitro and on bone formation and resorptionin vivoRésuméLa liaison du pyrophosphate inorganique (PPi) et de deux diphosphonates, l’éthane-1-hydroxy-1,1-diphosphonate disodique (EHDP) et le dichlorométhylène diphosphonate disodique (Cl2MDP), sur l’hydroxyapatite a été étudiée. Le PPi est lié plus fortement que l’EHDP, tandis que le Cl2MDP se lie le moins fortement. La liaison de ces trois substances s’accompagne d’une libération d’orthophosphate dans la solution et d’une adsorption de calcium par les cristaux. Il existe une compétition entre l’EHDP et le PPi; la capacié de l’EHDP à déplacer le PPi est plus grande que celle du PPi à déplacer l’EHDP. L’analyse des résultats à l’aide des fonctions de Scatchard suggère que la liaison n’est pas limitée à une catégorie de sites de liaison ayant une affinité constante et que l’affinité du premier site de liaison est plus grande pour l’EHDP que pour le PPi et plus grande pour le PPi que pour le Cl2MDP. Ces résultats sont en accord avec des résultats précédents concernant l’effet de ces composés sur la formation et la dissolution de cristaux d’apatitein vitro et la formation et la résorption osseusein vivo.ZusammenfassungDie Bindung von anorganischem Pyrophosphat (PPi) und zwei Diphosphonaten—Dinatrium-Äthan-1-hydroxy-1,1-diphosphonat (EHDP) und Dinatrium-Dichloromethylen-Diphosphonat (Cl2MDP)—an Hydroxyapatit wurde untersucht. Die Bindung ist am stärksten bei PPi, schwächer bei EHDP und am schwächsten bei Cl2MDP. Bei der Bindung aller drei Substanzen erfolgt gleichzeitig eine Abgabe von Orthophosphat in die Lösung und eine Aufnahme von Calcium durch die Kristalle. Es besteht eine Kompetition zwischen EHDP und PPi, wobei EHDP stärker ist im Verdrängen von PPi als umgekehrt. Eine Analyse mit Scatchard-Kurven weist darauf hin, daß die Bindung nicht auf eine Klasse von Stellen mit konstanter Affinität beschränkt ist und daß die Affinität der ersten Bindungsstelle die Reihenfolge EHDP>PPi>Cl2MDP hat. Die Resultate stimmen gut überein mit früheren Befunden über die Wirkung dieser Verbindungen auf die Bildung und Auflösung von Apatitkristallenin vitro und auf die Knochenbildung und-resorptionin vivo.

Characterization and cloning of the E11 antigen, a marker expressed by Rat Osteoblasts and Osteocytes

A new marker for cells of the osteoblastic lineage was identified by raising monoclonal antibodies against an immortalized rat osteoblastic cell line. Among the different antibodies one was selected which, on tissue sections, strongly reacts with osteoblasts, preosteocytes, and osteocytes. This antibody, designated E11, recognizes an antigen localized at the cell surface. The cDNA encoding the E11 antigen was cloned from a cDNA library prepared from ROS 17/2.8 cells, using a eukaryotic expression system. The E11 cDNA sequence revealed homology with the murine OTS-8/gp38 sequence. In situ hybridization confirmed that E11 mRNA expression in bone is restricted to osteoblasts and osteocytes. The tissue specificity of the E11 expression was studied by immunohistochemistry and Northern blot analysis. Apart from bone, E11-positive cells were also found in lung: namely, the alveolar cells of type I. Epithelial cells of the choroid plexus and endothelial cells of lymphatic vessels were also labeled with mAb E11. These results were confirmed by Northern blot, as the 1.8 kb E11 mRNA transcript was detected in bone and also in lung, brain, and skin. In conclusion, we describe a novel osteoblastic product which is expressed by mature osteoblasts and newly formed osteocytes.

Bisphosphonates : Preclinical aspects and use in osteoporosis

The bisphosphonates are synthetic compounds characterized by a P-C-P bond. They have a strong affinity to calcium phosphates and hence to bone mineral. In vitro they inhibit both formation and dissolution of the latter. Many of the bisphosphonates inhibit bone resorption, the newest compounds being 10,000 times more active than etidronate, the first bisphosphonate described. The antiresorbing effect is cell mediated, partly by a direct action on the osteoclasts, partly through the osteoblasts, which produce an inhibitor of osteoclastic recruitment. When given in large amounts, some bisphosphonates can also inhibit normal and ectopic mineralization through a physical-chemical inhibition of crystal growth. In the growing rat the inhibition of resorption is accompanied by an increase in intestinal absorption and an increased balance of calcium. Bisphosphonates also prevent various types of experimental osteoporosis, such as after immobilization, ovariectomy, orchidectomy, administration of corticosteroids, or low calcium diet. The P-C-P bond of the bisphosphonates is completely resistant to enzymatic hydrolysis. The bisphosphonates studied up to now, such as etidronate, clodronate, pamidronate, and alendronate, are absorbed, stored, and excreted unaltered. The intestinal absorption of the bisphosphonates is low, between 1% or less and 10% of the amount ingested. The newer bisphosphonates are at the lower end of the scale. The absorption diminishes when the compounds are given with food, especially in the presence of calcium. Bisphosphonates are rapidly cleared from plasma, 20%-80% being deposited in bone and the remainder excreted in the urine. In bone, they deposit at sites of mineralization as well as under the osteoclasts. In contrast to plasma, the half-life in bone is very long, partially as long as the half-life of the bone in which they are deposited. In humans, bisphosphonates are used successfully in diseases with increased bone turnover, such as Pagets disease, tumoural bone disease, as well as in osteoporosis. Various bisphosphonates, such as alendronate, clodronate, etidronate, ibandronate, pamidronate, and tiludronate, have been investigated in osteoporosis. All inhibit bone loss in postmenopausal women and increase bone mass. Furthermore, bisphosphonates are also effective in preventing bone loss both in corticosteroid-treated and in immobilized patients. The effect on the rate of fractures has recently been proven for alendronate. In humans, the adverse effects depend upon the compound and the amount given. For etidronate, practically the only adverse effect is an inhibition of mineralization. The aminoderivatives induce for a period of 2-3 days a syndrome with pyrexia, which shows a similitude with an acute phase reaction. The more potent compounds can induce gastrointestinal disturbances, sometimes oesophagitis, when given orally. Bisphosphonates are an important addition to the therapeutic possibilities in the prevention and treatment of osteoporosis.

Murine osteoblastlike cells and the osteogenic cell MC3T3-E1 release a macrophage colony-stimulating activity in culture

SummaryThe osteoclast may be of hematopoietic lineage and as such its development could be regulated by colony-stimulating factors. Since there is much interest as to whether osteoblasts influence bone resorption, we examined whether bone cells produce colony-stimulating activity. Both cells isolated from neonatal calvaria and the osteogenic cell MC3T3-E1 were found to constitutively release a colony-stimulating activity possessing characteristics of a macrophage colony-stimulating factor, as determined by basic biochemical purification and by identity of colonies induced in cultures of bone marrow cells. Release could be increased by the presence of the bone-resorbing agents lipopolysaccharide and 1,25 dihydroxyvitamin D3. We conclude that the osteoblast may contribute to both the processes of osteoclast formation and of hematopoiesis through the secretion of colony-stimulating activity into the adjacent bone marrow.

Expression of collagen, osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture

SummaryRat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of β-glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55±1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10-3 M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56°C with two slopes of inhibition. On SDS-PAGE, apparent molecular weight of ALP showed one band at 116,000 daltons (d) when extracted at cell confluence and two bands at 116,000 and 140,000 d when extracted at the 15th day of culture. 32P-labeled subunit of the enzyme migrated as one band at 75,000 d. Sialic acid content was demonstrated by neuraminidase treatment either on the dimeric form or on the 32P-labeled subunit. These data indicate that ALP expressed in this culture is bone specific. The results of the present study show that this mineralizing rat osteoblastic cell culture system synthesizes collagen type I, V, and traces of type III, osteocalcin, and bone ALP isoenzyme. Medium osteocalcin was detected during nodule formation and increased during mineralization. Increase in ALP activity as well as the presence of an additional form of ALP occurred in the mineralization phase. Therefore, this culture may be a useful model for studying the functions of bone-specific proteins during the process of mineralization.

Phosphate transport by rat renal brush border membrane vesicles: Influence of dietary phosphate, thyroparathyroidectomy, and 1,25-dihydroxyvitamin D3

In the present work we have investigated whether the changes in the renal handling of inorganic phosphate (Pi) induced by 1) dietary Pi, 2) removal of parathyroid glands and 3) 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], are associated with alterations in the Na-dependent Pi uptake by brush border membrane vesicles (BBMV) isolated from renal cortex. Shamoperated (SHAM) or thyroparathyroidectomized (TPTX) rats treated or not with 26 pmol/day of 1,25 (OH)2D3i.p. were fed low (0.2%) or high (1.2%)P diet for 7 days. The results showed that in SHAM, TPTX and TPTX+1,25 (OH)2D3 the Pi uptake by BBMV was greater after low than high Pi diet. It was greater in TPTX than in SHAM counterparts fed either diets. In TPTX fed low or high Pi diet 1,25 (OH)2D3 decreased the Pi uptake to the level observed in SHAM. A striking parallelism was found between variations in Pi uptake by BBMV and in the tubular Pi reabsorption of the whole kidney. The Na-dependent glucose, the mannitol uptake by BBMV, and the alkaline phosphatase activity in cortical homogenates and BBMV were not affected by the various treatments. Thus, dietary Pi, chronic TPTX and 1,25 (OH)2D3 appear to specifically affect the Na-dependent Pi transport system bound to the brush border membranes of renal cortical tubules. The alterations observed at this membrane level could account, at least in part, for the changes induced by these three factors on the overall tubular reabsorption of Pi.

Effect of insulin-like growth factor on collagen and glycosaminoglycan synthesis by rabbit articular chondrocytes in culture

Rabbit articular chondrocytes cultured in the presence of insulin-like growth factor I (IGF I) increased significantly the synthesis of both collagen and glycosaminoglycans. The increase in the ratio of35SO 4 2− to [3H]glucosamine observed in glycosaminoglycans synthesized in the presence of IGF I seems to indicate that IGF I affects sulphation and synthesis of these polyanionic macromolecules to a different extent.

Can Bisphosphonates Be Given to Patients with Fractures

One of the frequently asked questions concerning patients treated with bisphosphonates is whether individuals who recently have sustained a fracture should take inhibitors of bone resorption such as these. This argument is relevant because many of the patients treated with these drugs do have fractures, such as patients with osteoporosis, Paget’s disease, tumor bone disease, and more recently, osteogenesis imperfecta.

Transforming growth factor-β reduces the phenotypic expression of osteoblastic MP3T3-E1 cells in monolayer culture

Abstract Transforming growth factor beta (TGF-β) regulates cell growth and differentiation. Since it is abundant in bone, we have studied the effect of the polypeptide upon the growth and phenotypic expression of murine osteoblastic cells in monolayer culture. Its actions were compared to those of epidermal growth factor (EGF), another hormonally active polypeptide known to alter bone cell function. Picogram amounts of TGF-β were found to inhibit the growth and phenotype (alkaline phosphatase and cAMP response to parathyroid hormone) of the clonal nontransformed MC3T3-E1 osteoblastic cell line. EGF also inhibited phenotypic expression, although at higher (nanogram) concentrations, but stimulated cell growth. The low concentration of TGF-β required to inhibit growth and phenotype of osteoblastic cells together with its abundance in bone suggest that TGF-β may be an important regulator of bone cell function.