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PubMed1990

Streaming potential of intact wet bone.

The conversion of mechanical loads to bioelectrical signals in bone have been suggested to control repair and remodeling. These signals in wet bone are attributed to the electrokinetic behavior where mechanical forces cause electrical signals due to motion of an ion carrying extracellular fluid in the bone matrix (streaming potentials). Streaming potential experiments were performed on control and chemically treated intact wet bone plugs in aphosphate and phosphate buffers to examine the contribution of bone constituents to the electrokinetic behavior of bone tissue. Data indicate that the organic constituents of bone dominate streaming potentials. Slopes of streaming potential vs pressure are related to the electrokinetic (zeta) potential. The slopes should be analyzed in the low pressure region where data is mainly linear. Comparisons of estimated zeta potentials from streaming potentials with existing data obtained by particle electrophoresis showed similar trends.

Animals↗
PubMed2006

Human degenerative valve disease is associated with up-regulation of low-density lipoprotein receptor-related protein 5 receptor-mediated bone formation.

OBJECTIVES: The goal of this research was to define the cellular mechanisms involved in myxomatous mitral valve disease and calcific aortic valve disease and to redefine the term degenerative valve disease in terms of an active cellular biology. BACKGROUND: "Degenerative" valvular heart disease is the primary cause of regurgitant and stenotic valvular lesion in the U.S. However, the signaling pathways are not known. We hypothesize that valve degeneration occurs due to an osteoblastic differentiation process mediated by the low-density lipoprotein receptor-related protein 5 (Lrp5) signaling pathway to cause valve thickening. METHODS: We examined human diseased valves: myxomatous mitral valves (n = 23), calcified tricuspid aortic valves (n = 27), calcified bicuspid aortic valves (n = 23), and control tissue from mitral and aortic valves (n = 40). The valves were examined by reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry for signaling markers important in osteoblast differentiation: Sox9 and Cbfa1 (transcription factors for osteoblast differentiation); Lrp5 and Wnt3 (osteoblast differentiation signaling marker), osteopontin and osteocalcin (osteoblast endochrondral bone matrix proteins), and proliferating cell nuclear antigen (a marker of cell proliferation). Cartilage development and bone formation was measured by Alcian blue stain and Alizarin red stain. Computed Scano MicroCT-40 (Bassersdorf, Switzerland) analysis measured calcium burden. RESULTS: Low-density lipoprotein receptor-related protein 5, osteocalcin, and other osteochrondrogenic differentiation markers were increased in the calcified aortic valves by protein and gene expression (p > 0.001). Sox9, Lrp5 receptor, and osteocalcin were increased in myxomatous mitral valves by protein and gene expression (p > 0.001). MicroCT was positive in the calcified aortic valves and negative in the myxomatous mitral valves. CONCLUSIONS: The mechanism of valvular heart disease involves an endochondral bone process that is expressed as cartilage in the mitral valves and bone in the aortic valves. Up-regulation of the Lrp5 pathway may play a role in the mechanism for valvular heart disease.

Aged↗
PubMed1992

Effects of non-steroidal anti-inflammatory drugs on demineralized bone-induced bone formation.

The inhibitory effect of some non-steroidal anti-inflammatory drugs (NSAIDs) on bone resorption is well documented. To explore the effect of NSAIDs on bone formation, we investigated the time course and dose/response characteristics of treatment with various non-steroidal anti-inflammatory drugs (NSAIDs) on ectopic bone formation induced by demineralized bone matrix (DBM) in the rat model. Using biochemical assays, both inhibitory and stimulatory effects on bone formation were found in rats treated with NSAIDs prior to DBM implantation depending on the type and amount of drug administered. There appears to be an enhancement of bone formation for acetaminophen (50 mg/kg), acetylsalicylic acid (50 mg/kg), and ibuprofen (50 mg/kg). Indomethacin (4 mg/kg) and piroxicam (4 mg/kg) had inhibitory effects. Flurbiprofen, on the other hand, did not appear to affect bone formation significantly. In contrast, there was no effect of NSAIDs on bone formation in rats treated with the drugs after implantation of the DBM. It appears that the time course of the drug administration is critical, suggesting that early events in bone formation may be modulated by arachidonic acid metabolites.

Acetaminophen↗
PubMed1993

Changes in the expression of insulin-like growth factor II/mannose-6-phosphate receptor during endochondral bone development.

Endochondral bone development can be induced by subcutaneous implantation of demineralized bone matrix (DBM) in rats. We used this in vivo model to study the relationship between endochondral bone formation and expression of IGF-II/M-6-P receptor, a multifunctional protein which binds not only IGF-II, but also lysosomal enzyme bearing mannose-6-phosphate motif. We found that IGF-II/M-6-P receptor was present in implants from day 1 to day 21; the highest levels were expressed on day 11 during bone differentiation. IGF-II/M-6-P receptor mRNA content was highest on day 9. We conclude from these data that IGF-II/M-6-P receptor expression is developmentally regulated during endochondral bone formation. This regulation occurs in part at the level of IGF-II/M-6-P receptor mRNA. The relatively high level of IGF-II/M-6-P receptor during ossification suggests that this receptor might play a role in bone formation and remodeling.

Animals↗
PubMed1994

Effect of 1 alpha-vitamin D3 on bone strength and composition in growing rats with and without corticosteroid treatment.

The effects of 1 alpha-vitamin D3 were studied for 6 months in 2-month-old male and female rats on a moderately low calcium diet with or without low-dose prednisolone treatment. Both cortical bone mechanical and biochemical properties were examined. Femoral bone specimens were subjected to torsional loading tests. With age, bone strength and stiffness increased in both sexes, accompanied by an increased degree of mineralization (bone ash and calcium concentrations). During growth, strength and stiffness increased more in male than in female rats. When 1 alpha-vitamin D3 (0.5 micrograms/kg/day) was given alone, bone mechanical competence improved significantly whereas insulin-like growth factor-I (IGF-I) and calcium concentrations in the bone matrix were significantly reduced. Treatment with low-dose prednisolone (0.5 mg/kg/day) alone did not influence bone mechanical properties compared with intact control rats (without prednisolone) although a significant reduction in calcium concentration and an increased phosphorus concentration were measured. A combined therapy with prednisolone and 1 alpha-vitamin D3 significantly increased bone strength, toughness, and stiffness compared with control bones. Both mineralization degree (ash and calcium concentration) and IGF-I concentration were decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Biologic principles of bone induction.

This article provides a concise review of bone induction. Bone induction by demineralized bone matrix is a multistep cascade. The purification and elucidation of the chemistry of osteogens will improve bone grafting methods.

Alkaline Phosphatase↗
PubMed1986

Control of repair in articular tissues and the development of osteoarthritis.

Bones from different mammalian species, including man, contain a low molecular weight extractable activity which stimulates proteoglycan synthesis by connective tissue cells in culture ("matrigenin" activity). Partial purification of matrigenin activity has been achieved from bovine bone. The activity shows a strong tendency for reversible physical associations in low ionic strength solutions and binds tightly to collagenous and other bone matrix proteins. It is speculated that the pathophysiological role of matrigenins may be to initiate repair during injury to bone. Chronic repair reactions are more likely to occur at sites of repeated anatomical stress at the ligamentous, tendon and synovial attachments at the ends of bones, in the subchondral bone and in the articular cartilage regions abbuting to subchondral bone. At these sites, repair reactions may be broadly viewed as the development of osteophytes and fibrocartilage and the progression of subchondral bone sclerosis. Repeated anatomical stress leads to the activation of extracellular, small molecular weight factors, which in turn, stimulate the biosynthesis of matrix macromolecules that are an important feature of these repair reactions of osteoarthritis.

Animals↗
PubMed2000

Integrins and signaling in osteoclast function.

Integrins are heterodimeric adhesion receptors that mediate cell-matrix and cell-cell interactions. Osteoclasts highly express the alphavbeta3 integrin, which binds to a variety of extracellular matrix proteins including vitronectin, osteopontin and bone sialoprotein. RGD-containing peptides, RGD-mimetics and alphavbeta3 blocking antibodies inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in osteoclast function. RGD-containing peptides were shown to raise cytosolic calcium in osteoclasts. Furthermore, several signaling and adaptor molecules were found to be involved in alphavbeta3 integrin-dependent signaling pathways, including phosphatidylinositol 3-kinase, c-Src, PYK2 and p130(cas). In addition, cytoskeletal molecules such as paxillin, vinculin, gelsolin and F-actin are recruited to adhesion contacts upon integrin activation. Many of these molecules signaling and cytoskeletal localize to the sealing zone of actively resorbing osteoclasts, suggesting that they play a role in linking the adhesion of osteoclasts to the bone matrix with the cytoskeletal organization and the polarization and activation of these cells for bone resorption.

Animals↗
PubMed1977

Osteocyte ultrastructure in renal osteodystrophy.

The ultrastructure of the osteocyte has been studied in 80 needle biopsies from the iliac crest of uremic subjects with renal osteodystrophy. Different types of osteocytes were present in the osseous trabeculae. Those recognizable in completely uncalcified osteoid tissue looked like normal osteocytes, even though the matrix was not mineralized. Those present in hypomineralized areas showed enlarged and irregular lacunae when examined under the light microscope; under the electron microscope these osteolytic-like changes were not evident and were found to have been produced by defective calcification of the perilacunar matrix. Osteocytes placed in matrix whose mineralization was normal were often surrounded by a border of crystals protruding side-to-side from the bone matrix into the lacunar space. Other osteocytes were placed in unusually wide lacunae. They showed evidence of osteolytic activity, chiefly consisting of irregularity of the lacunar wall, presence of flocculent, granular and filamentous material in the pericellular space, and calcification of mitochondria. Degenerating and degenerate osteocytes were also recognizable.

Adolescent↗
PubMed1988

Bone resorption by isolated chick osteoclasts in culture is stimulated by murine spleen cell supernatant fluids (osteoclast-activating factor) and inhibited by calcitonin and prostaglandin E2.

The question of whether any of the agents known to activate bone resorption in vivo or in organ cultures acts directly on the osteoclast or via intermediate target cells that secondarily secrete locally paracrine factors is important for our understanding of bone remodeling. In an attempt to clarify this issue for some of the agents, we have taken advantage of the recent progress in obtaining and culturing relatively pure populations of osteoclasts. We performed an in vitro bone-resorbing assay in which isolated and partially purified chick osteoclasts were cultured on devitalized, paired and standardized bone disks prepared from rat calvaria prelabeled with both 45Ca and 3H-proline. Some of the isolated osteoclasts attached to the devitalized bone matrix, formed a ruffled border, and acidified the bone-resorbing compartment that they established with the matrix, thereby indicating that they resorbed bone in a physiologic manner. Salmon calcitonin added to these cultures (0.3 U/ml = 60 ng/ml) and prostaglandin E2 (PGE2) (10(-6) M) inhibited both basal and stimulated 45Ca and 3H-proline release. Neither parathyroid hormone (PTH) 1-34 (1 U/ml), 1,25-(OH)2-D3 (10(-8) and 10(-9) M), nor interleukin 1 (IL-1) (purified from P388D1 macrophage culture supernatant fluids or recombinant murine IL-1-alpha) (100 ng/ml) stimulated bone resorption in these cultures. In contrast, supernatant fluids from concanavalin A (Con-A)-activated murine spleen cell cultures (murine osteoclast-activating factor; OAF) consistently and significantly induced a 3- to 5-fold stimulation of bone resorption in this system.

Animals↗
PubMed1998

Spatiotemporal assessment of fetal bovine osteoblast culture differentiation indicates a role for BSP in promoting differentiation.

Fetal bovine mandible-derived osteoblasts were cultured for the purpose of obtaining a spatiotemporal assessment of bone matrix protein expression during in vitro differentiation. The results obtained from electron microscopic, immunohistological, biochemical, and molecular biological analyses indicated that these primary cultured osteoblasts produce an abundant extracellular matrix which mineralizes during a 14-day culture period. During this process, a restricted, spatiotemporal pattern of bone sialoprotein expression was indicated by immunohistological and molecular evaluations. To test the possibility that bone sialoprotein promoted the continued morphodifferentiation of osteoblastic cells, cultures were grown in the presence of anti-bone sialoprotein antibodies known to interfere with cell-bone sialoprotein attachment. Compared with cultures grown in the presence of normal rabbit serum (1:150), cultures grown in the media containing anti-bone sialoprotein antibody (1:150) failed to mineralize as demonstrated by von Kossa staining and failed to express osteocalcin and osteopontin as shown by the reverse transcription polymerase chain reaction. These results contribute to the growing evidence that bone sialoprotein is an important determinant of osteoblast differentiation and bone formation. Matrix protein-cell interactions may be examined using this spatiotemporally defined model.

Animals↗
PubMed1984

Effects of iron overload on bone remodeling in pigs.

For study of the effects of an iron overload on bone remodeling, 5 control pigs were compared with 5 pigs given a total dose of 10.8 g of parenteral iron in 36 days. Treated pigs developed an iron tissue overload demonstrated by a marked increase in bone and liver iron. Except for a modest increase in SGOT, there was no biochemical or histologic sign of liver damage. Serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were unchanged in the treated pigs. There was no accumulation of iron in the parathyroid glands and the serum immunoreactive parathyroid hormone level was unchanged in the treated animals. Bone histomorphometry after double tetracycline labeling showed that in the treated pigs osteoblast cell surfaces, double and total labeled surfaces, appositional rate, and formation at tissue level were significantly decreased, and reversal surfaces were increased. Mineralization was not impaired because the osteoid thickness was unchanged. From the morphometric measurements it was concluded that osteoblast recruitment and the collagen synthesis rate were decreased. Mean wall thickness, which indicates the amount of bone synthesized, was also lowered. In contrast, the osteoclastic resorption surfaces and the depth of lacunae resulting from osteoclast resorption were unchanged by treatment. Despite this imbalance between formation and resorption, trabecular bone mass estimated on trabecular bone volume and bone ash was unchanged after 36 days' treatment. Perls' stain revealed that iron deposits were present in osteoblast and osteoclast cells and also inside the bone matrix, because there was a linear deposit along the trabecular surfaces, cement line, and osteoid-mineralized bone interface. Therefore, because treatment induced no modification of the major humoral regulators of bone metabolism, it is suggested that iron, which was present in bone cells and matrix, could play a role in bone remodeling.

Alanine Transaminase↗
PubMed2002

Ultrastructural distribution of osteoadherin in rat bone shows a pattern similar to that of bone sialoprotein.

Osteoadherin (OSAD) is a keratan sulfate proteoglycan recently isolated from bovine and rat bone. Based on results obtained from in vitro experiments, the protein was shown to bind osteoblasts via the integrin receptor alpha v beta 3. Due to OSAD's capacity to bind hydroxyapatite crystals, a role for the protein in the mineralization process has also been suggested. To test these hypotheses in an in vivo model, the ultrastructural localization of OSAD in bone, tibial (metaphyses and diaphyses), and calvarial samples from normal 10 to 12-day-old rats were examined by immunohistochemical techniques at the ultrastructural level. In addition to the qualitative studies, quantitative measurements of OSAD marker density were performed in relevant compartments. Immunolabeling for OSAD was located to the mineralized bone matrix, with highest concentration of marker at the border between bone and cartilage remnants in the metaphyseal trabeculi. Intracellular labeling was low and no systemic accumulation of OSAD markers was observed at the cell-matrix interface. The observed distribution pattern of OSAD is strikingly similar to that of bone sialoprotein (BSP), confirmed by double labeling. The results of the current study support a role for OSAD in the mineralization process. In this process BSP is assumed to be a nucleator of hydroxyapatite crystals, and OSAD could work in concert with BSP to regulate nucleation. However, the mechanisms involved remain to be elucidated.

Animals↗
PubMedDate not supplied

Hyaluronate production by the inner ear during otic capsule and perilymphatic space formation.

Otosclerosis has been hypothesized to result from a disorder of the extracellular matrix of the cartilaginous rests present in the adult temporal bone. Matrix relationship to bone formation and remodeling, as well as the fact that the pathogenesis of otosclerosis is expressed by the action of both of these processes, strongly suggests that more knowledge is needed about the process of otic capsule development. In pursuit of this goal, otic complexes were explanted from mouse embryos that ranged in age from 10.5 to 16 days old and were then exposed to 3H-glucosamine (50 microCi/ml) for 6 hours in vitro. Total labeled glycosaminoglycans (GAGs) and labeled hyaluronate content of each age group of otic explants were measured, and the results were compared to a developmental series of the otic regions of whole embryos stained with either toluidine blue or alcian blue. Increases in the synthesis of the total GAGs were observed on embryonic day 11 and for a prolonged period extending from gestation day 13.5 through day 16. The first increase of GAGs occurred at the initiation of metachromasia and positive staining by toluidine blue of the region of aggregated periotic mesenchyme cells that form the otic capsule. The second increase in GAGs was correlated with chondrification of the capsule. Hyaluronate production revealed a different pattern. Synthesis of hyaluronate exhibits peaks at 10.5, 12.5, and for an extended period of from 13.5 to 14.5 days of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Matrix proteins.

The past decade has witnessed a major increase in our knowledge of bone matrix proteins. These advances stem from investigations of genetically altered mice and the phenotypic analysis of the consequences of the deletion of these proteins. Surprisingly, however, some genes expected to yield considerable phenotypes because of their specificity for bone--for example, osteocalcin--exhibit only subtle changes. A picture is beginning to emerge that suggests important interactions between matrix proteins and other molecules in the bone environment, cytokines, cell surface receptors, growth factors, and other matrix proteins. Future studies require investigation of downstream events of protein ablation and require investment in microarray and proteomics. Double and triple deletions of relevant bone proteins may provide further insight into the function of these important components of bone and lead to new therapeutics for the treatment of osteoporosis and other metabolic bone diseases.

Animals↗
PubMed1980

Sialic acids in bone demineralised in organic solvents.

The sialic acid content of bovine shin bone, demineralised by a new process in ethanol-trimetrylammonium EDTA, was measured and compared with that of the original whole bone. New variants of the direct Ehrlich procedure, as well as the periodate-thiobarbituric acid assay, were employed. About 75% of the sialic acid remains in the bone matrix from ethanolic demineralisation, and the PAS reaction is correspondingly much more intense, compared with matrix from aqueous EDTA demineralisation.

Animals↗
PubMed1995

Injectable bone marrow preparations to stimulate osteogenic repair.

The great versatility of bone marrow transplants based on stem cell activity has been demonstrated successfully for a variety of previously untreatable hemopoietic conditions. Autologous bone marrow delivered by percutaneous injection or by direct transplant as a composite graft also has proven effective for osteogenic stimulation in a series of 100 skeletal healing problems, including delayed unions and nonunions of fractures, arthrodeses, and bone defects. The efficiency of marrow to form bone can be increased by a number of methods, including differential centrifugation and composite grafts of marrow with demineralized bone matrix and other carriers or stimulatory factors.

Animals↗