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PubMed1993

Initiation and promotion of bone differentiation by bone morphogenetic proteins.

The presence of growth and differentiation factors in bone has been demonstrated by subcutaneous implantation of demineralized bone matrix that initiates new cartilage and bone morphogenesis. The genes for bone morphogenetic proteins (BMPs) have been cloned and expressed. Recombinant BMPs induce endochondral bone formation in vivo. The multistep sequential developmental cascade consists of chemotaxis, mitosis, and differentiation of cartilage and bone. The pleiotropic response has been well characterized. BMPs stimulate osteogenic and chondrogenic phenotypes. Natural bovine osteogenin (BMP-3) and recombinant BMP-4 are equipotent in chemotaxis, limb bud chondrogenesis, cartilage maintenance, and in vivo bone induction. There are multiple isoforms of BMPs, raising the biologic relevance of the redundancy. The mode of action and second messengers are not clear. BMPs appear to have cognate receptors as demonstrated by iodinated BMP-2B (BMP-4). Other novel members of the BMP family include osteogenic protein 1 (BMP-7) and osteogenic protein 2 (BMP-8). Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily and include three distinct subfamilies: BMP-2, BMP-3, and BMP-7. Native BMP-3 and recombinant BMP-4 bind type IV collagen of the basement membrane. This novel connection may be the long elusive mechanistic explanation for the requirement of angiogenesis and vascular invasion for bone morphogenesis. BMPs may have a role in fracture repair, periodontal regeneration, and alveolar ridge augmentation.

Animals↗
PubMed1994

Brittle bones in spontaneously diabetic female rats cannot be predicted by bone mineral measurements: studies in diabetic and ovariectomized rats.

Spontaneously diabetic BB rats were sham operated (SO) or ovariectomized (OVX) within days after onset and studied after 4, 8, and 12 weeks. Analyses included histomorphometry of proximal tibial metaphyses, biochemical analyses of humeri, DXA analyses, and biomechanical testing of femora. In SO diabetic rats, no osteoblasts, osteoid tissue, or osteoclasts were present on the trabecular bone surface, but trabecular bone volume (TBV) remained normal compared with control BB rats. The concentration of IGF-I per dry weight of humerus was decreased after 12 weeks of diabetes, whereas the concentrations of calcium and osteocalcin did not change. DXA analysis showed normal bone mineral density (BMD) at both diaphyseal and metaphyseal femoral areas. On biomechanical testing, angular deformation, energy absorption, and torsional strength of the femora were decreased after 8-12 weeks of diabetes, but stiffness was normal. Ovariectomy in diabetic rats caused a decrease in femoral BMD especially at the metaphysis, and there was a trend toward decreased TBV in the tibial metaphysis; TBV loss was less marked than in control OVX rats, however. The increase in BMD at the femoral diaphysis, measured after 12 weeks of OVX in control rats, was absent in diabetic rats. Multiple-regression analysis indicated that the presence of diabetes but not ovariectomy, weight, and mineral content correlated with decreased energy absorption, angular deformation, and strength of the femora. The data infer that the (near) absence of unmineralized bone matrix in severely diabetic rats alters bone microarchitecture and ultimately results in brittle bones, which is not predicted by BMC or BMD measurements.

Absorptiometry, Photon↗
PubMed1994

Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures.

Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either alpha-MEM with 2% heat-inactivated horse serum alone (alpha) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal alpha medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase-positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 microgram/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 +/- 14.2) than in GM-CSF alone (53.3 +/- 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption.

Acid Phosphatase↗
PubMed1991

Effects of indomethacin on demineralized bone-induced heterotopic ossification in the rat.

Indomethacin inhibits bone formation when treatment is initiated before the implantation of demineralized bone matrix (DBM). For the inhibition of bone induction to occur, indomethacin treatment had to be initiated 6 h or more before implantation of DBM. Initiating the drug treatment at or after the time of DBM implantation had no effects on the amounts of new bone formed. The inhibition by indomethacin is dose related over a range between 0.04 and 4 mg/kg body weight. Recovered day-1 DBM implants, transplanted into indomethacin pre- and posttreated syngeneic rats, formed bone at the same rate as controls did. However, recovered day-1 DBM implants lyophilized before transplantation showed decreased bone formation but significant dystrophic calcification as judged by a lower alkaline phosphatase activity and an elevated calcium content.

Alkaline Phosphatase↗
PubMed1996

Effects of lead on growth plate chondrocyte phenotype.

Lead toxicity is a major public health problem in the United States. The skeleton serves as the major reservoir for ingested lead, where it is incorporated into bone matrix during calcification. While lead in bone has been considered inactive, mounting clinical and epidemiological data has shown a strong correlation between lead exposure and adverse effects on stature in children. These epidemiologic data suggest a direct effect of lead on skeletal development, but whether it reflects a systemic effect, a specific effect on osteoblasts, or an effect on the epiphyseal growth plate is as yet unclear. This study examined the effects of lead on parameters of cartilage biology in isolated chondrocytes. Changes in growth plate chondrocyte phenotype were assessed utilizing an established avian growth plate chondrocyte model. Low, sublethal doses of lead caused specific and significant effects on a number of important markers of growth plate chondrocyte phenotype, including suppression of alkaline phosphatase and both type II and type X collagen expression at the protein and mRNA levels, and a decrease in thymidine incorporation. In contrast, proteoglycan synthesis was stimulated relative to controls in lead-treated cultures, suggesting that the alterations in collagen and DNA synthesis and alkaline phosphatase activity are not due to cytotoxity. The data demonstrate important regulatory effects of lead on growth plate chondrocytes in cell culture and suggest an inhibitory effect on the process of endochondral bone formation. The growth plate may be one of the key target tissues accounting for the adverse effects of chronic lead exposure on skeletal development.

Animals↗
PubMed1979

Relationship between blood flow and radiostrontium uptake in the healing bone fracture.

The healing of a diaphyseal tibia fracture was followed in the rat. Callus formation, blood flow and Sr-85 uptake were assessed by a ratio comparison of the fractured to the contralateral side. No correlation was found between blood flow and Sr-85 deposition in the callus and the adjacent bone of the same extremity. It is concluded that the deposition of a radiopharmaceutical in bone is primarily related to the presence of calcifiable organic bone matrix and only secondarily to the bone formation rate and to variations in the local blood flow.

Animals↗
PubMed1985

The influence of stress and strain in the early development of shaft bones. An experimental study on the chick embryo tibia.

In chick embryos from stage 23 to stage 27 the whole presumptive zeugopod - or its pre-axial (tibial) portion only - was proximodistally and dorsoventrally inverted by turning it 180 degrees round the anteroposterior axis of the limb bud. Development of the reoriented blastema of the tibia was consistently retarded and variously reduced: this skeletal piece appeared shorter and relatively thicker than the controlateral normal tibia. Chondrification, progress of differentiation of the cartilaginous model, onset and gradual spreading of the ossification processes were considerably delayed. Often the diminutive tibia underwent a degree of bending or angulation of up to 90 degrees - 100 degrees in the sagittal plane. In these bent tibiae - obviously developing under abnormal conditions of intrinsic and extrinsic mechanical stresses - cell hypertrophy appeared greatly retarded or hindered in sites of the diaphysial cartilaginous core which were presumably subjected to strong longitudinal compression. No rigorous temporal and topographical relationships were observed between chondrocyte hypertrophy and onset of perichondral osteogenesis. Apparently, a direct contact between hypertrophic cartilage and perichondrial cells was not strictly required to prompt osteogenesis; this process, in fact, often involved areas of the perichondrium enveloping parvicellular cartilage. Radial pressures exerted by the over-stretched outer layer of the perichondrium, or periosteum, on the subjacent prospective osteogenous layer reduced or prevented the deposition of bone. Conversely, radial stretching of the inner layer of the perichondrium, or periosteum, considerably enhanced cell proliferation, blood vessel formation, differentiation of osteoblasts and formation of bone matrix.

Animals↗
PubMed1979

Bone modelling processes at the endosteal surface of human femora. Scanning electron microscopical studies in normal bone and in renal osteodystrophy.

In femoral bone of 10 adult patients without bone disease and of 15 patients with secondary hyperparathyroidism, thy after nonmineralised organic material had been removed from the endosteal surface by sodium hypochlorite. This technique permits one to analyse the effects of past osteoblastic and osteoclastic activities. Im normal bone, the endosteal envelope shows a highly ordered texture: The main part of the inner surface is represented by fully mineralised smooth surfaces without evidence of apposition or resorption (so called neutral surfaces). In apposition areas, collagen fibers are still incompletely mineralised. Ordered mineral deposits are observed, consisting of spindleshaped calcospherites of uniform size. The resorption areas are sharply delimited. The resorption layer shows a small difference of level with respect to the surrounding neutral surface. Resorption areas consist of numerous lacunae with a smooth bottom. Individual lacunae are encircled by shallow ridges which run almost perpendicularly to the main direction of collagen fibers that have been exposed by resorption. These findings suggest that in normal bone osteoclasts act as a corrdinated group of cells. The direction of advance of the resorption area is to some extent influenced by the collagen pattern of bone. In patients with secondary hyperparathyroidism, domain formation of the endosteal surface can no longer be recognized. The size and shape of caleospherites are extremely heterogeneous, a finding interpreted as evidence of formation of woven bone. Resorption areas are irregularly determined and often resemble worm-eaten wood. The planes of resorption vary in direction and depth and in general resorption cavities penetrate deeper than in normal bone. These findings point to loss of coordinated cell action under the influence of hyperparathyroidism. The observations suggest that in hyperparathyroidism endosteal cells do not respond to local factors which influence endosteal cell activities in modelling processes of normal bone. Such local factors consist of the pattern of collagen andlamellar organisation on one hand and mechanical forces presumably via pizo-electrical potentials, on the other. In hyperparathyroidism the interdependence between bone matrix texture and spatial orientation of bone surface lining cell activities is lost.

Adult↗
PubMed2000

Mechanical loading stimulates differentiation of periodontal osteoblasts in a mouse osteoinduction model: effect on type I collagen and alkaline phosphatase genes.

The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hybridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This increase reflected enhanced gene expression in individual osteoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semiquantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteoblasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically-induced differentiation of osteoblasts. (4) osteogenic mechanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix-depositing osteoblasts located on the bone surface and within the osteoid layer.

Alkaline Phosphatase↗
PubMed2001

Increased levels of somatostatin in rat ankles with adjuvant arthritis.

Levels of somatostatin were investigated in the ankles and spinal cords of rats suffering from acute and chronic adjuvant arthritis. As measured by radioimmunoassay, somatostatin showed significantly higher concentrations only in chronic arthritic ankles. No significant difference was observed in somatostatin levels between the spinal cords of normal and arthritic groups. Using immunohistochemical labeling and electron microscopy, we observed increased somatostatin labeling in the mature bone matrix, monocytes, and polymorphonuclear cells of bone marrow and macrophage-like synovial cells of chronically arthritic rats. Understanding the mechanism(s) which lead to increased somatostatin in chronic arthritic joints may result in more effective treatment methods.

Animals↗
PubMed2000

Development and fine structure of pharyngeal replacement teeth in juvenile zebrafish (Danio rerio) (Teleostei, Cyprinidae).

Teeth are commonly used model systems for the study of epithelial-mesenchymal interactions during organogenesis. We describe here the ultrastructural characteristics of developing pharyngeal replacement teeth in juvenile zebrafish, an increasingly important model organism for vertebrate development. Replacement teeth develop in close association with the dental organ of a functional tooth. Morphogenesis is well advanced prior to the start of cytodifferentiation. Fibrillar enameloid matrix is formed first, followed by the deposition of predentine. Initial mineralization of the enameloid proceeds quickly; maturation involves the presence of ruffled-bordered ameloblasts. Dentine mineralization is inotropic and is mediated by matrix vesicles. Woven-fibred attachment bone matrix is deposited before completion of dentine mineralization. Eruption of fully ankylosed teeth is a fast process and may involve degenerative changes in the pharyngeal epithelium. Mononucleated osteoclasts and clastic cells located in the pulp cavity intervene in tooth resorption prior to shedding. Structural differences with larval, first-generation zebrafish teeth include the presence of dentinal tubules and the absence of an electron-dense covering membrane. Part of these differences may relate to size differences of the teeth. Others, like the site of the replacement tooth bud, suggest that initiation may take place in already committed epithelium from the first initiation event in the larval stage.

Amelogenesis↗
PubMed1991

Isolation of a cathepsin B-encoding cDNA from murine osteogenic cells.

Cathepsin B-encoding cDNA (CTSB) clones have been isolated from a lambda gt10 library of a murine osteosarcoma by differential screening during a search for genes which are typically expressed during osteogenic differentiation in mouse mandibular condyles in vitro. Sequencing of the CTSB 3' end revealed that the isolated sequence contained an 825-bp 3'-noncoding region, the polyadenylation signal and the poly(A) tail. The enhanced CTSB expression during the early stages of the enchondral ossification-like process in mandibular condyles in vitro suggests that CTSB participates in the degradation of cartilage matrix prior to the synthesis of bone matrix proteins.

Animals↗
PubMed1989

Chemotactic response of osteoblast-like cells to transforming growth factor beta.

Transforming growth factor beta (TGF-beta) was tested for its ability to stimulate a chemotactic response in two clonal rat osteosarcoma (ROS) cell lines, 17/2 and 25/1. TGF-beta stimulated dose-dependent chemotaxis in both cell lines. In serum-containing media, maximal response was seen at a concentration of 500 fg (10(-15)g)/mL for the ROS 17/2 cells and 25 fg/mL for the ROS 25/1 cells. In serum-free media, the maximal chemotactic response to TGF-beta occurred at 5 fg/mL for both the ROS 17/2 and 25/1 cells. TGF-beta was not mitogenic at these dosages. The results indicate that TGF-beta could act as a chemoattractant for osteogenic cells in both demineralized bone matrix induced osteogenesis and in normal bone remodeling.

Animals↗
PubMed1992

Inhaled beclomethasone decreases serum osteocalcin in postmenopausal asthmatic women.

There are very few data as yet to quantify the effect of inhaled corticosteroids on bone metabolism, although the use of these drugs as a first-line treatment in bronchial asthma has widened. We determined the effect of three dose levels (200, 1000, 2000 micrograms/day, three weeks each) of inhaled beclomethasone on specific characteristics of bone metabolism in nine postmenopausal women with new asthma without any previous corticosteroid therapy. Significant decrease was noted in the mean serum morning osteocalcin concentration between the baseline and after nine weeks of beclomethasone (from 4.4 to 3.1 micrograms/l, p = 0.005). Significant increase in serum total and ionised calcium was found, although the parameters measuring bone resorption itself did not change. The results show that especially high-dose inhaled beclomethasone decreases serum osteocalcin in post-menopausal asthmatic women. Further studies are needed to assess the effects of inhaled beclomethasone, both on the ability of the osteoblasts to form bone matrix and on the density of bone during a longer treatment period on inhaled corticosteroids.

Administration, Inhalation↗
PubMed2005

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier.

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.

Aged↗
PubMed2006

Age-related changes in lamin A/C expression in the osteoarticular system: laminopathies as a potential new aging mechanism.

BACKGROUND: Mutations in lamin A/C have been described as associated to severe changes in bone and joints. In this study we hypothesize that the expression of lamin A/C may play a role in the pathogenesis of age-related diseases in the osteoarticular system. METHODOLOGY: C57BL/6 young and old mice (4 months; n=10 and 24 months; n=10) were sacrificed. Limbs were isolated for histopathological and Western blot analysis. The proportion of cells (osteoblasts and chondrocytes) positive for lamin A/C was quantified by immunohistochemistry. Lamin B1 was used as control. Finally, lamin A/C expression in bone marrow cells was quantified by Western blot. RESULTS: A significant reduction in lamin A/C was found in osteoblasts of old as compared to young mice (42% versus 76%, p<0.001). Interestingly, lamin A/C but not lamin B1 expression was found in bone matrix with higher levels in young bone. Additionally, a significant reduction in the number of lamin A/C expressing chondrocytes was seen in old mice as compared to young mice (32% versus 84%, p<0.001). Finally, a reduction in lamin A/C expression was found in bone marrow cells obtained from old mice as compared to young mice. CONCLUSION: This is the first assessment of the age-related changes in lamin A/C expression in the osteoarticular system. We conclude that with aging there is a reduction in lamin A/C expression which could have a significance on osteoarticular cells function and viability.

Aging↗
PubMed2003

Urinary excretion of deoxypyridinoline increases after gastrointestinal surgery.

OBJECTIVES: We investigated the effect of gastrointestinal surgery on bone metabolism with special reference to nutrition status and the systemic inflammatory response (SIR). METHODS: We assessed bone resorption by measuring the urinary excretion of deoxypyridinoline (D-Pyr), a specific marker that reflects the amount of degraded collagen. Twenty patients who underwent gastrectomy or colectomy were enrolled in this study. Daily energy intake, nitrogen, calcium, and phosphate balances, and urinary D-Pyr were examined preoperatively and for 14 days after the operation. The nutritional risk index and prealbumin were measured for nutrition assessment, and SIR was evaluated daily based on scorings of body temperature, pulse rate, respiratory rate, and white blood cell number according to our criteria. RESULTS: Urinary D-Pyr excretion had already increased on postoperative day 1 and continued to increase until postoperative day 14. The amounts of postoperative urinary excretion of D-Pyr correlated positively with the SIR scores and the amount of urinary excretion of cortisol, one of the stress-response hormones, and inversely with pre- and postoperative nutritional risk indices. In addition, the patients who experienced complications during the postoperative period excreted larger amounts of D-Pyr. CONCLUSIONS: Because the amount of excreted D-Pyr reflects the loss of the bone matrix, these results may indicate that bone resorption increases after gastrointestinal surgery. The extent of resorption was parallel to the degree of SIR and nutrition status.

Adult↗
PubMed1991

Biosynthesis of bone proteins by fetal porcine calvariae in vitro. Rapid association of sulfated sialoproteins (secreted phosphoprotein-1 and bone sialoprotein) and chondroitin sulfate proteoglycan (CS-PGIII) with bone mineral.

To study the biosynthesis of bone proteins, fragments of fetal porcine calvariae were cultured in the presence of 50 micrograms/ml ascorbate and 10 mM beta-glycerophosphate and individual cultures labeled for either 4 h or 48 h with [35S]-methionine, Na2[35SO4], Na3[32PO4] or [14C]-glycine plus [14C]-proline. The radiolabeled proteins in tissue extracts were obtained by sequential extraction with 4 M GuHCl (G1-extract), 0.5 M EDTA (E-extract), and again with 4 M GuHCl (G2-extract) and analyzed together with the radiolabeled proteins secreted into the medium. SPP-1 (secreted phosphoprotein 1, osteopontin) was the major non-collagenous protein deposited into the bone matrix, with lesser amounts of BSP (bone sialoprotein), osteocalcin and chondroitin sulfate proteoglycans (CS-PG II and CS-PG III). SPP-1 was also the major phosphorylated protein and was recovered, together with several fragmented forms, almost entirely in the demineralizing extracts. Moreover, approximately one-half of the [35SO4] incorporated into E-extract proteins was present in SPP-1, the remainder being incorporated into PGs with smaller amounts associated with BSP. Over 65% of the [35SO4] in the proteoglycans of the demineralizing extracts was recovered in the small CS-PG III with less than 35% in CS-PG II, the bone homologue of DS-PG II (decorin). In contrast, CS-PG II was the predominant small proteoglycan in culture media and in guanidine extracts. Some sulfated BSP was also observed in guanidine extracts and small amounts appeared to bind to collagen. Radiolabeled SPARC (osteonectin), a prominent protein of fetal porcine bone, was not detected in the mineralized bone tissues but was prominent in the culture medium. These results demonstrate that following secretion, the major proteins expressed by osteoblastic cells are initially incorporated into different tissue compartments, with most of the sulfated sialoproteins and CS-PG III associating rapidly with the hydroxyapatite crystals. The initial distribution of these proteins is of importance in the evaluation of their role in bone formation and mineralization.

Animals↗

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