Oenology: red wine procyanidins and vascular health.

Regular, moderate consumption of red wine is linked to a reduced risk of coronary heart disease and to lower overall mortality, but the relative contribution of wine's alcohol and polyphenol components to these effects is unclear. Here we identify procyanidins as the principal vasoactive polyphenols in red wine and show that they are present at higher concentrations in wines from areas of southwestern France and Sardinia, where traditional production methods ensure that these compounds are efficiently extracted during vinification. These regions also happen to be associated with increased longevity in the population.

Thrombocytopenia in the toothless (osteopetrotic) rat and its rescue by treatment with colony-stimulating factor-1.

Osteopetrosis in toothless (tl) rats is characterized by reductions in bone resorption, osteoclasts, and macrophages, resistance to cure by bone marrow transplantation, and skeletal improvement after treatment with colony-stimulating factor-1 (CSF-1). Reductions in skeletal osteocalcin tl rats, together with the recent demonstration of osteocalcin expression in platelets and its possible role in bone turnover, prompted us to examine whether this rat mutation is associated with altered platelet numbers. Our prediction of a thrombocytopenia was confirmed by examination of tl rats, in which a profound reduction (32%) in platelets was accompanied by a significant elevation (62%) in megakaryocytes (MKC) compared to normal littermates. Light and transmission electron microscopy confirmed increases in both number and size of MKC in mutants without morphologic abnormalities of circulating platelets. CSF-1 treatment (10(6) U/48 hours for 10 days) of mutants restored platelet numbers to those found normal littermates and increased osteoclasts and the frequency of MKC in numbers. Preliminary studies of another mutation the rat, osteopetrosis (op), revealed a similar reduction (33%) in platelets. These data demonstrate the coexistence of osteopetrosis and thrombocytopenia in two osteopetrotic rat mutations and an increase in osteoclasts and platelets in one mutation after CSF-1 treatment. Together, these data suggest a potential functional interaction of MKC and osteoclasts in bone turnover.

The mechanisms and mediators of tooth eruption--models for developmental biologists.

Tooth eruption is a localized process in the jaws which exhibits precise timing and bilateral symmetry. It involves resorption and formation of bone on opposite sides of the erupting tooth and these activities depend on the dental follicle, a thin connective tissue investment of the developing and erupting tooth. Biochemical studies have shown that during eruption cells, proteins and enzymes change in the dental follicle and several growth factors and proteins known to accelerate or retard eruption have been identified. This review discusses these aspects of tooth eruption and proposes testable hypotheses and strategies that can make studies of tooth eruption new experimental opportunities for developmental biologists.

Colony-stimulating factor-1 (CSF-1) rescues osteoblast attachment, survival and sorting of beta-actin mRNA in the toothless (tl-osteopetrotic) mutation in the rat.

We have shown that in the osteopetrotic rat mutation toothless (tl) osteoblasts are absent from older bone surfaces in mutants and that mutant osteoblasts in vivo lack the prominent stress fiber bundles polarized along bone surfaces in osteoblasts from normal littermates. Our recent data demonstrate that in normal osteoblasts in vitro beta- and gamma-actin mRNAs have different, characteristic intracellular distributions and that tl (mutant) osteoblasts fail to differentially sort these mRNAs. Because bone resorption and formation are highly interdependent and injections of CSF-1, a growth factor, increase bone resorption and growth in tl rats, we examined the effects of CSF-1 treatment on osteoblast survival and ultrastructure in vivo and ability to sort actin mRNAs in vitro. Neonatal CSF-1 treatment of mutants restores osteoblasts on older bone surfaces, normalizes the intracellular distribution of stress fibers in osteoblasts in vivo and promotes normal sorting of beta-actin mRNA in mutant osteoblasts in vitro without normalizing gamma-actin distribution. These data suggest the beta- and gamma-actin mRNAs in osteoblasts are sorted by different mechanisms and that the differential sorting of beta-actin mRNA is related to the characteristic polarization of stress fibers in osteoblasts and their survival on bone surfaces. This experimental system can be used to explore the relationships and regulation of these aspects of cell and tissue biology.

Endochondral bone formation in toothless (osteopetrotic) rats: failures of chondrocyte patterning and type X collagen expression.

The pacemaker of endochondral bone growth is cell division and hypertrophy of chondrocytes. The developmental stages of chondrocytes, characterized by the expression of collagen types II and X, are arranged in arrays across the growth zone. Mutations in collagen II and X genes as well as the absence of their gene products lead to different, altered patterns of chondrocyte stages which remain aligned across the growth plate (GP). Here we analyze GP of rats bearing the mutation toothless (tl) which, apart from bone defects, develop a progressive, severe chondrodystrophy during postnatal weeks 3 to 6. Mutant GP exhibited disorganized, non-aligned chondrocytes and mineralized metaphyseal bone but without cartilage mineralization or cartilaginous extensions into the metaphysis. Expression of mRNA coding for collagen types II (Col II) and X (Col X) was examined in the tibial GP by in situ hybridization. Mutant rats at 2 weeks exhibited Col II RNA expression and some hypertrophied chondrocytes (HC) but no Col X RNA was detected. By 3rd week, HC had largely disappeared from the central part of the mutant GP and Col II RNA expression was present but weak and in 2 separate bands. Peripherally the GP contained HC but without Col X RNA expression. This abnormal pattern was exacerbated by the fourth week. Bone mineralized but cartilage in the GP did not. These data suggest that the tl mutation involves a regulatory function for chondrocyte maturation, including Col X RNA synthesis and mineralization, and that the GP abnormalities are related to the Col X deficiency. The differences in patterning in the tl rat GP compared to direct Col X mutations may be explained by compensatory effects.

Evidence that the rat osteopetrotic mutation toothless (tl) is not in the TNFSF11 (TRANCE, RANKL, ODF, OPGL) gene.

The toothless (tl) osteopetrotic mutation in the rat affects an osteoblast-derived factor that is required for normal osteoclast differentiation. Although the genetic locus remains unknown, the phenotypic impact of the tl mutation on multiple systems has been well characterized. Some of its actions are similar to tumornecrosis factor superfamily member 11(TNFSF11; also called TRANCE, RANKL, ODF and OPGL) null mice. TNFSF11 is a recently described member of the tumor necrosis factor superfamily which, when expressed by activated T cells, enhances the survival of antigen-presenting dendritic cells, and when expressed by osteoblasts, promotes the differentiation and activation of osteoclasts. The skeletal similarities between tl rats and TNFSF11(-/-) mice include 1) profound osteoclastopenia (TNFSF11-null mice, 0% and tl rats 0-1% of normal); 2) persistent, non-resolving osteopetrosis that results from 3) a defect not in the osteoclast lineage itself, but in an osteoblast-derived, osteoclastogenic signal; and 4) a severe chondrodysplasia of the growth plates of long bones not seen in other osteopetrotic mutations. The latter includes thickening of the growth plate with age, disorganization of chondrocyte columns, and disturbances of chondrocyte maturation. These striking similarities prompted us to undertake studies to rule in or out a TNFSF11 mutation in the tl rat. We looked for expression of TNFSF11 mRNA in tl long bones and found it to be over-expressed and of the correct size. We also tested TNFSF11 protein function in the tl rat. This was shown to be normal by flow cytometry experiments in which activated, spleen-derived T-cells from tl rats exhibited normal receptor binding competence, as measured by a recombinant receptor assay. We also found that tl rats develop histologically normal mesenteric and peripheral lymph nodes, which are absent from TNFSF11-null mice. Next, we found that injections of recombinant TNFSF11, which restores bone resorption in null mice, had no therapeutic effect in tl rats. Finally, gene mapping studies using co-segregation of polymorphic markers excluded the chromosomal region containing the TNFSF11 gene as harboring the mutation responsible for the tl phenotype. We conclude that, despite substantial phenotypic similarities to TNFSF11(-/-) mice, the tl rat mutation is not in the TNFSF11 locus, and that its identification must await the results of further studies.

Bafilomycin A1 inhibits bone resorption and tooth eruption in vivo.

It has been shown that a specific inhibitor of vacuolar H(+)-ATPases, bafilomycin A1, inhibits bone resorption by isolated chicken osteoclasts by blocking the proton pump in the ruffled border membrane. We report here the effects of bafilomycin A1 on bone resorption in vivo. Using a cannulated osmotic minipump delivery system, we infused bafilomycin locally to the eruption pathway of permanent premolars of beagle dogs. We used pit formation by osteoclasts in vitro to estimate the concentrations and heat stability of bafilomycin to be used in vivo. In this model, osteoclasts were cultured on thin bone slices, in which they form pits indicative of resorption. After 2 weeks preincubation at 37 degrees C, bafilomycin concentrations of 10(-6) and 10(-7) M but not 10(-8) M completely inhibited the resorptive activity of cultured osteoclasts, and the two larger doses were chosen for use in vivo. Local delivery of 10(-6) M bafilomycin to the eruption pathway of the fourth permanent mandibular premolar during mideruption inhibited tooth eruption by blocking bone resorption as assayed by radiography, light microscopy, and scanning electron microscopy. Bafilomycin at 10(-7) M had similar but less intensive effects. Moreover, osteoclasts in the alveolar bone of crypts treated with 10(-7) M bafilomycin A1 stained very weakly for tartrate-resistant acid phosphatase. The effect of bafilomycin on bone resorption was shown to be very local, and no side effects of treatment with bafilomycin were observed in adjacent teeth or the behavior of dogs. We report here, for the first time, inhibition of tooth eruption caused by inhibited bone resorption using bafilomycin A1 in vivo.

The giant cells recruited by subcutaneous implants of mineralized bone particles and slices in rabbits are not osteoclasts.

We have compared structural and functional characteristics of native osteoclasts and the multinucleated giant cells (MNGC) recruited by subcutaneous implants of mineralized bone particles and slices in normal rabbits. Weekly evaluation of the implants for 5 weeks showed distinct differences between MNGC and osteoclasts in the host with respect to morphology and the ability to stain for tartrate-resistant acid phosphatase and acid ATPase. An osteoclast-specific monoclonal antibody bound strongly to osteoclasts but not MNGC. Ground bone slices similarly implanted were surrounded by MNGC but did not show resorption pits by scanning electron microscopy. These data show that the MNGC recruited to subcutaneous implants of mineralized bone particles and slices lack the enzymatic, cell surface, and functional features of osteoclasts.

Tumor necrosis factor modulates apoptosis of monocytes in areas of developmentally regulated bone remodeling.

Tooth eruption is characterized by spatially segregated bone resorption along the path of eruption and bone formation in the opposite direction. Monocyte recruitment occurs in two distinct peaks in both areas of resorption and formation. Without such recruitment tooth eruption does not occur. The signals that regulate this recruitment are thought to involve the expression of cytokines and chemokines. One such cytokine is tumor necrosis factor (TNF), which can affect monocyte recruitment through the induction of chemokines and adhesion molecules and increase their lifespan by acting as antiapoptotic cell survival signals. We examined the latter by studying mice with targeted deletions of TNF receptors p55 and p75 (TNFRp55/p75). The results indicate that mice that lack functional TNF receptors have a significantly reduced number of monocytes in the apical area associated with bone formation. The reduced number of monocytes in this area can be accounted for by an increase in apoptosis in TNFRp55-/-/p75-/-. In contrast, the number of monocytes, the rate of monocyte apoptosis, and the formation of osteoclasts in the occlusal area associated with bone resorption occurred independently of TNF activity. These results suggest that TNF receptor signaling can affect tooth eruption by acting as a monocyte survival signal in some but not all areas of bone undergoing developmentally regulated remodeling.

Calcitonin receptor binding as a marker of osteoclast heterogeneity in osteopetrotic rodents.

We have employed a radioautographic technique to examine in vivo receptor binding of calcitonin to osteoclasts in four rodent mutants with osteopetrosis. 125I-Labeled calcitonin was injected intravenously alone or with excess unlabeled calcitonin to osteopetrotic (op/op), osteosclerotic (oc/oc), and microphthalmic (mi/mi) mice and to incisor absent (ia/ia) rats. Similar experiments were performed simultaneously in phenotypically normal littermates. Specific binding of calcitonin to receptors on osteoclasts and osteoclast morphology were then examined by light and electron microscope radioautography. Calcitonin binding was increased in mi/mi mice, where osteoclasts were abundant but reduced in size, and was also increased in op/op mice in association with an undulated and redundant osteoclast cell membrane. Binding of the hormone was markedly diminished on osteoclasts of oc/oc mice and ia/ia rats. Thus, in these rodent models of osteopetrosis all of which manifest reduced skeletal remodeling and share a recessive pattern of inheritance, considerable heterogeneity of osteoclast characteristics was demonstrable. Although calcitonin may play no primary pathogenetic role in most forms of this disease, calcitonin receptor binding is a morphological and functional marker of osteoclasts that can be used in assessing the pathophysiology of disorders of bone remodeling.

Evaluation of urinary pyridinium crosslink excretion as a marker of bone resorption in the rat.

The aim of this study was to evaluate the value of the urinary excretion of the pyridinium crosslinks, pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), as markers of bone resorption in the rat. The excretion of the crosslinks was compared with that of urinary [3H]tetracycline ([3H]TC) excretion from chronically [3H]TC-prelabeled animals, a technique established to monitor bone resorption in the rat. Bone resorption was modulated by Ca restriction, infusion of PTH, thyroparathyroidectomy, and administration of different bisphosphonates. Furthermore, the urinary crosslinks were assessed in three different osteopetrotic mutations in the rat. We found a delayed response of Pyr and D-Pyr excretion to acute changes in bone resorption compared with [3H]TC excretion. This delay was 1 day after Ca restriction and longer after other treatments, such as PTH administration or bisphosphonate treatment, with which it was more than 3 weeks. In contrast, chronic states with stimulation or inhibition of bone resorption showed similar changes in excretion of the urinary crosslinks and [3H]TC, except after PTH administration. The excretion of the crosslinks was greatly reduced in osteopetrotic rats (op/op, tl/tl, and ia/ia) and increased to normal levels in tl/tl rats after stimulation of bone resorption by M-CSF administration. These results suggest that, in rats, urinary excretion of the pyridinium crosslinks reflects bone resorption in chronic but not always in acute conditions. The cause of this discrepancy is still unclear.

Coexistence of reduced function of natural killer cells and osteoclasts in two distinct osteopetrotic mutations in the rat.

Recent evidence suggesting that immune cells and their products (cytokines) play an important role in the regulation of skeletal development and function, particularly of the osteoclast, implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders. The mammalian osteopetroses are a pathogenetically heterogeneous group of skeletal disorders characterized by skeletal sclerosis resulting from reduced osteoclast-mediated bone resorption. Using a 51Cr-release microcytotoxicity assay we demonstrated that splenic natural killer (NK) cell activity was significantly reduced in two distinctly different osteopetrotic mutations in the rat, osteopetrosis (op) and toothless (tl). To determine whether this reduction in NK cell-mediated cytotoxicity is caused by decreased cell number and/or function in these osteopetrotic mutants, we quantitated NK cells by analyzing mononuclear cell suspensions labeled for two-color fluorescence with OX8 and OX19 monoclonal antibodies in a fluorescence-activated cell sorter. Flow cytometry of these double-labeled cells revealed that the percentage of NK cells (OX8+/OX19- subset) in op and tl spleens was not significantly different from that of normal spleens. These results suggest that NK cells in these osteopetrotic mutants are functionally defective. Thus aberrations in osteoclast and NK cell function coexist in these mutations, and their developmental relationships deserve further study.

Treatment of congenital osteopetrosis in the rabbit with high-dose 1,25-dihydroxyvitamin D.

Osteopetrosis is a congenital metabolic bone disease characterized by skeletal sclerosis resulting from defective osteoclast-mediated bone resorption. Osteopetrosis has been described in several animal species (mouse, rat, and rabbit) and in children. Bone marrow transplantation, originally shown to reverse the skeletal sclerosis in some animal mutations, has been effective in curing osteopetrosis in some children. Unfortunately, not all children with osteopetrosis are candidates for or respond to bone marrow transplantation. Recent studies have shown that several animal mutations and some children inheriting osteopetrosis have significantly elevated serum levels of 1,25-(OH)2D. Based on the possibility that there may be a resistance to 1,25-(OH)2D, high-dose calcitriol therapy has been used to treat some children and stimulated some parameters of resorption. In this study, we have examined the effects of high-dose calcitriol therapy on various serum and skeletal parameters in the osteopetrotic rabbit. Mutant rabbits and normal littermates were given continuous infusions of calcitriol via subcutaneously implanted osmotic minipumps for 2 weeks at a dose of 0.5, 2.5, or 25 micrograms/kg/per day. Untreated mutant rabbits are hypocalcemic and hypophosphatemic in the presence of elevated serum 1,25-(OH)2 levels in comparison with their normal littermates. Calcitriol infusions resulted in dose-dependent increases in circulating 1,25-(OH)2D levels in both normal and mutant rabbits. However, evaluation of other serum parameters and the skeletal response demonstrated significant differences between osteopetrotic and normal rabbits. At the highest dose, normal animals rapidly became hypercalcemic and osteoporotic, accompanied by weight loss and a failure to thrive; mutants remained hypocalcemic and osteopetrotic but did not exhibit the deleterious physical effects seen in treated normal littermates. Although the number of osteoclasts increased in both mutants and normals, osteoclast phenotype in the former remained abnormal. These data indicate that although very high levels of circulating 1,25-(OH)2D were achieved in osteopetrotic mutants, activation of osteoclast-mediated bone resorption with subsequent improvement of skeletal sclerosis was not observed.

Mineral density and bone strength are dissociated in long bones of rat osteopetrotic mutations.

Bone mineral density (BMD) and mechanical strength generally show strong positive correlations. However, osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. We have evaluated mechanical strength and mineral density in three osteopetrotic mutations in the rat (incisors-absent [ia/ia], osteopetrosis [op/op], and toothless [tl/tl]) to test the hypothesis that reduced bone resorption in one or more of these mutations results in weaker bones in the presence of greater mineral density and skeletal mass. Peripheral quantitative computed tomography (pQCT) was used to analyze BMD and cross-sectional geometry in the tibial diaphysis and metaphysis as well as the femoral diaphysis and femoral neck. The bending breaking force of tibial and femoral midshafts was obtained using the three-point bending test and femoral neck strength was tested by axial loading. Osteopetrotic mutants were significantly smaller than their normal littermates (NLMs) in each stock. The pQCT analysis showed that BMD and bone mineral content (BMC) were higher than or equal to NLMs in all skeletal sites measured in the osteopetrotic mutants. However, the mechanical breaking force was equal to or lower than their NLMs in all sites. The cross-sectional structure of long bone shafts was markedly different in osteopetrotic mutants, having a thin cortex and a medullary area filled with primary trabecular bone. These results indicate that osteopetrotic mutations in the rat increase bone density and decrease bone strength. The tibial diaphysis was significantly weaker in tl/tl and ia/ia mutants and the tibial metaphysis showed the greatest increase in BMD in all mutants. These data are another illustration that an increased BMD does not necessarily lead to stronger bones.

Normalization of mineral homeostasis after reversal of osteopetrosis.

Whether a radiographic and histologic cure of osteopetrosis includes normalization of mineral homeostasis remains unknown. Thus, we explored the extent of defective mineral metabolism in the microphthalmic (mi/mi) mouse before and after cure. Under basal conditions mi mutants exhibit normocalcemia, hypophosphatemia, and elevated renal 25-hydroxyvitamin D-1-hydroxylase activity. However, administration of PTHrP (3 micrograms/h x 24 h) further stimulated enzyme activity in mi mutants with active disease, to a level no different than that in treated normals. Serum phosphorus levels also declined in mi/mi mice following PTHrP, suggesting a normal renal response to this hormone. In contrast, failure to suppress enzyme function in mi/mi mice following prolonged calcitriol infusion indicates that the observed enhancement of 1,25-dihydroxyvitamin D production occurred secondary to autonomous parathyroid function and/or nonparathyroid hormone-related stimuli. Although an increased fractional excretion and decreased tubular reabsorption of phosphate were demonstrated in mi/mi mice, serum PTH levels were no different in mi mutants compared with normal littermates. Following skeletal cure, the mi/mi mice surprisingly display normal serum phosphorus levels and renal enzyme activity. Moreover, treatment restored normal responsiveness to calcitriol suppression and maintained normal PTHrP responsiveness of enzyme activity. These data indicate that the cure of osteopetrosis in the mi mutant is universal and includes normalization of serum phosphorus and renal 25-hydroxyvitamin D-1-hydroxylase. Furthermore, these data suggest that phosphate depletion of unknown origin is the likely cause of elevated enzyme activity in this murine osteopetrotic mutant.

Auditory ossicle abnormalities and hearing loss in the toothless (osteopetrotic) mutation in the rat and their improvement after treatment with colony-stimulating factor-1.

Osteopetrosis describes a group of skeletal metabolic diseases of heterogeneous etiology and varied severity that produces a generalized accumulation of skeletal mass, the result of reduced bone resorption. Inherited in a variety of species including humans, the most severe forms are lethal. Among common features are progressive blindness and deafness of controversial etiologies for which there are no universally effective treatments. We have studied the auditory responsiveness and auditory ossicle quantitative histomorphology and temporal bone vasculature in the toothless (tl) rat, a lethal osteopetrotic mutation with few osteoclasts, very low bone turnover, and limited angiogenesis in the axial skeleton. Compared with normal littermates, 3-week-old mutants showed significantly reduced auditory responsiveness, a hearing loss due to abnormalities in both form and tissue composition of the stapes, and little capillary sprouting in the vascular bed of the temporal bone. Treatment of mutants with colony-stimulating factor 1 (CSF-1), known to greatly reduce sclerosis in the axial skeleton, significantly improved hearing, stapedial form and tissue composition, and angiogenesis in the temporal bone. In normal rats, the stapes consisted of 89.3% bone, 9.1% mineralized cartilage, and 0.8% porosity. In osteopetrotic rats, the stapes consisted of 48.3% bone, 35.9% mineralized cartilage, and 15.9% porosity, while after CSF-1 treatment, the bone content increased to 55.2%, cartilage was decreased to 21.7%, and porosity increased to 23.0%, respectively. This is the first demonstration of an auditory abnormality in an osteopetrotic animal mutation and shows that the hearing loss in tl rats can be significantly improved following treatment with CSF-1.

Osteopetrosis in the rat: coexistence of reductions in osteocalcin and bone resorption.

Osteocalcin, one of the vitamin K-dependent bone proteins, has recently been implicated in bone resorption. To explore this hypothesis, bone and serum osteocalcin were measured in three different osteopetrotic rat mutations characterized by reduced bone resorption. These three mutations (ia/ia, tl/tl, and op/op) exhibit heterogeneity with respect to osteoclast number and activity and response to being cured by bone marrow transplantation. Calvarial bone osteocalcin was present in normal amounts, but difficult to extract, in ia/ia rats that have increased numbers of inactive osteoclasts. Bone osteocalcin was greatly decreased in op/op (53-60% of control) and tl/tl (64-73% of control) osteopetrotic rats, in which osteoclasts are both reduced in number and inactive. These decreases in osteocalcin levels in bone coexist with elevated serum levels of osteocalcin in all three mutations. Since osteocalcin synthesis is known to be stimulated by 1,25(OH)2D3, the increase in serum osteocalcin may be a reflection of the elevated blood levels of 1,25(OH)2D3 known to occur in each of these mutations. These findings indicate that the composition of osteopetrotic bone is abnormal with respect to osteocalcin in the two rat osteopetrotic mutations showing decreased osteoclast numbers. Considered together with the emerging evidence that the extracellular matrix in many developing tissues plays a role in cell recruitment and differentiation, these data suggest that osteocalcin abnormalities may be a contributing factor to the spectrum of osteoclast aberrations in osteopetrosis.

Parathyroid hormone stimulation of adenylate cyclase activity and lactic acid accumulation in calvaria of osteopetrotic (ia) rats.

We have examined the effect of parathyroid hormone (PTH) on the adenylate cyclase activity of newborn osteopetrotic rat calvaria, to study a possible molecular basis for the reduced response of this mutant to PTH. Phenotypically normal littermates served as controls. We also measured the effect of PTH on kidney adenylate cyclase activity and on lactic acid accumulation in short term cultures of calvaria. PTH stimulated calvarial adenylate cyclase activity in a dose-dependent manner in both mutant rats and normal littermates. Lactic acid production was also enhanced by PTH, and no significant difference between mutants and normal littermates was observed. These findings indicate that the reduced response of the young osteopetrotic rats to PTH is not due to an absence of PTH receptors coupled to adenylate cyclase.

Identification and characterization of the genes encoding human and mouse osteoactivin.

Osteoactivin (OA) is more highly expressed in the bones of osteopetrotic mutant rats (op/op) than in those of their normal littermates and is the homologue of human nmb, a cDNA more highly expressed in melanoma-derived cell lines of low metastatic potential, and of mouse DC-HIL, which has been implicated in endothelial cell adhesion. The human OA gene is found on chromosome 7p15.1 and consists of 11 exons spanning 28.3 kb. Murine OA is encoded by a highly similar gene of 11 exons spanning 20.2 kb on mouse chromosome 6. Human OA uses the same transcriptional initiation site in both bone and kidney as was reported for melanoma cells. OA is expressed in primary human and mouse osteoblast cultures at all stages of differentiation, with increased levels observed concurrently with the expression of osteoblast phenotype markers. OA is also expressed in a wide variety of human and mouse tissues as determined by RT-PCR analysis. Immunohistochemical investigation of OA expression in late mouse embryonic development showed very high, cell-specific expression in the nervous system, basal layer of the skin, germinal cells of hair follicles, and in the forming nephrons of the kidney. Continuing investigation of the cell-specific expression of OA in bone as well as in other tissues will lead to a better understanding of its function in the development of these cell types.

Local stimulation of new bone formation by prostaglandin E1: quantitative histomorphometry and comparison of delivery by minipumps and controlled-release pellets.

E-series prostaglandins (PGE) given systematically or locally in vivo can have powerful anabolic effects on bone. The comparative dose-response relationships from PGE1 given by osmotic minipump or controlled-release pellet on periosteal and intracortical bone envelopes were determined. Graded doses of PGE1 were delivered by osmotic minipump (0.0 to 16.7 mg PGE1/week) or controlled-release pellet (0.0 to 16.7 mg PGE1/week) to the lateral mandibular surface of adult dogs (2-5 years old). PGE1 was given for three weeks and tissues were collected for histology and histomorphometry one week later. At sites of maximal response, there were dose-related increases in the periosteal surface with new bone formation, average new bone thickness, maximum thickness, and new bone area. Subperiosteal bone formation was greater for comparable doses when PGE1 was delivered by minipumps. The proliferation of new bone at those sites treated with the pellets was greatest with the 8.3 mg PGE1/week/three week treatment. Periosteal bone formation did not appear to be preceded by a resorption phase, indicating that PGE1 treatment stimulated bone modeling in the formation mode. The new bone consisted of woven bone, particularly at sites with high accretion rates, and primary lamellar bone. There were some dose-related changes in intracortical remodeling indices including increases in cortical porosity, single and double-labeled surface, numbers of formation, reversal and resorption osteons, radial closure rate, and surface- and volume-referent bone formation rates. Increases in the bone formation rates indicate that PGE1 increased the recruitment of osteoblasts. Increases in the mineral appositional rate, observed at a higher dose, indicates that PGE1 stimulated bone production at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)