摘要:

AbstractProliferation of osteoblast‐like MC3T3‐El cells was minimal in serum‐free Eagle's minimum essential medium (MEM) but was enhanced by about 3.5‐fold in serum‐free (alpha)‐modification of MEM ((alpha)‐MEM). By adding back each of the extra constituents present in (alpha)‐MEM to MEM, it was found that ascorbic acid was responsible for the sustained proliferation of MC3T3‐El cells without serum. Ascorbic acid also stimulated the synthesis of collagen and increased the hydroxyproline content of MC3T3‐El cell cultures markedly. Inhibitors of collagen synthesis, L‐azetidine‐2‐carboxylic acid,cis‐4‐hydroxyproline, and 3,4‐dehydroproline, almost completely eliminated the stimulatory effect of ascorbic acid on DNA synthesis of MC3T3‐El cells. The dose response of the effect of L‐azetidine‐2‐carboxylic acid on the hydroxyproline content closely paralleled that on DNA synthesis of MC3T3‐El cells. Furthermore, a 10 times higher concentration of proline, which competes with L‐azetidine‐2‐carboxylic acid for the incorporation into procollagen molecules, reversed the inhibition of DNA synthesis by L‐azetidine‐2‐carboxylic acid. These results are consistent with the assumption that the stimulatory effect of ascorbic acid on the proliferation of MC3T3‐El cells is mediated through its effect on the synthesis of collagen or some related protein. Furthermore, a fibronectin attachment peptide, GRGDTP, that competes with matrix proteins for specific binding to cell surface adhesion receptors also inhibited the stimulation of proliferation by ascorbic acid almost completely. It is suggested that ascorbic acid stimulates osteoblast proliferation through its effect on the synthesis of collagen or some related protein and that such a matrix protein interacts with cell surface adhesion receptors to cause the stimulation of proliferation in osteoblasts. The mechanism of how such a protein affects t

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060902

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have studied the incorporation of radioactive P (32P) into lipids of bovine parathyroid tissue under conditions of stimulated and inhibited hormone secretion. Utilizing low (0.5 mM) and high (3.0 mM) concentrations of calcium to regulate parathyroid hormone secretion, we initially found that the labeling of the cellular phospholipids with32P was greater in those tissues incubated in high‐calcium medium. Thin‐layer chromatography of lipid extracts prepared from tissue incubated in either low‐ or high‐calcium media revealed that the increased incorporation of32P (high or low) was localized primarily to two phospholipids. To determine whether the increases were due directly to the different calcium concentrations, the experiments were performed in media containing normal calcium concentrations (1.25 mM) and low (0.5) or high (3.0) magnesium concentrations to modulate hormone secretion. The results were identical to those obtained using low and high calcium, indicating that the increased32P incorporation was not an effect of high calcium but rather correlated with the inhibition of hormone secretion. The use of other secretagogues confirmed this correlation. The identity of the two phospholipids was established, by two‐dimensional thin‐layer chromatography, to be phosphatidylinositol (PI) and lysophosphatidylinositol (LPI). The correlation of increased32P incorporation with inhibition of secretion led us next to examine isolated secretory granules from tissues exposed to either high‐or low‐calcium conditions. Thin‐layer chromatography of granule lipid extracts yielded chromatograms containing PI and LPI, and the radioactivity of each was greater in the high‐calcium sample than in the low‐calcium sample. Our data suggest that a pathway for the synthesis and/or recycling of PI and LPI is present in secretory granules. Taken together, our observations suggest that phosphatidylinositol and lysophosphatidylinositol may play a role, either directly or indirectly, in

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060903

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractThe relationship between bone‐resorbing cells, assessed by the presence of tartrate‐resistant acid phosphatases (TRAP) and morphologic indices of bone resorption, was determined in 29 osteoporotic patients (14 postmenopausal females and 15 males) and 15 dialyzed patients. The number of TRAP‐positive cells per unit of cancellous bone area (N.Oc/B.Ar) was higher in dialyzed patients than in those with osteoporosis (16.8 (pminus) 15.3 versus 4.95 (pminus) 2.86,p<0.05). The amount of bone resorbed at the basic multicellular unit level was estimated by calculating eroded area containing TRAP cells per bone area (E.Ar+/BA). This novel parameter was similar in dialyzed and in osteoporotic patients (41,700 (pminus) 28,400 versus 32,300 (pminus) 24,600). In contrast, trabecular spacing (Tb.Sp) was identical in both metabolic bone diseases. Trabecular width (169 (pminus) 38 versus 127 (pminus) 32 (mu)m,p<0.05) and bone area were higher in dialyzed than in osteoporotic patients. N.Oc/B.Ar was significantly related to E.Ar+/BA in dialyzed (r= 0.76,p<0.05) but not in osteoporotic patients. Tb.Sp was significantly correlated to N.Oc/B.Ar and to the number of TRAP‐positive cell nuclei per B.Ar (r= 0.44,p<0.05) in osteoporotic but not in dialyzed patients. This last result shows that in overt osteoporosis with thin trabeculae, trabecular spacing is related to the number of resorbing cells. In contrast, the spacing of thick trabeculae in dialysis osteodystrophy is not dependent on the number of oste

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060904

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractWe observed that culture medium conditioned with fetal rat long bones contained peptides immunologically related to the parathyroid hormone‐related peptide of malignancy (PTHrP) and stimulated cyclic AMP production in canine renal cortical membranes. Because the adenylate cyclase stimulating activity (CSA) of the medium increased when bone resorption was stimulated, it was suspected that these peptides were stored in the matrix and released during the resorption process. In this work, we extracted the noncollagenous proteins of fetal rat long bones and found that the extract contained significant amounts of CSA. The biologic activity of the extract was abolished after trypsin digestion and eluted at 24 and 37 kD on filtration HPLC. The CSA of bone extract and of both HPLC peaks could be inhibited by 3–34 and 7–34 parathyroid hormone analogs. It was not blocked by an antiserum directed against the N‐terminal region of parathyroid hormone, but it was significantly inhibited after an overnight preincubation with an antiserum directed against the 1–11 fragment of PTHrP. One band migrating at 18 kD could be visualized after SDS‐PAGE electrophoresis of bone extract and immunoblotting with the anti‐PTHrP antiserum. We conclude that an adenylate cyclase stimulator immunologically similar to PTHrP is present in the matrix of fetal rat long bones. Adenylate cyclase stimulating peptides of lower molecular weight found in bone‐conditioned medium could be active fragments formed by proteolysis during the res

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060905

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractIn 28 patients with idiopathic or postmenopausal type 1 (spinal crush fracture) osteoporosis, resorption indices and dynamic measurements of trabecular bone formation based on in vivo tretracycline labeling in 7.5 mm transiliac biopsies have been compared with trends in radial cortical and trabecular bone density measured with computed tomography. Positive correlations were observed between trabecular bone density trends in the radius and indices of bone formation in the ilium. These were improved when one of the two resorption indices was included with a formation index in bivariate regressions. Marked interindividual variations in radial bone density trends were also seen in cortical bone. These correlated poorly with trends in trabecular bone. Weak negative relationships between cortical bone trends and indices relating to bone formation and resorption were observed, but a positive association was seen with single‐labeled surfaces on iliac trabeculae. If, as has been suggested, there are periodic variations in bone formation, the results suggest that axial and peripheral trabecular bone density trends are synchronized in osteoporosis, perhaps in response to systemic factors, such as circulating hormone

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060906

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractAqueous tissue processing and demineralization procedures may adversely affect the inorganic mineral phase of a calcified sample and, where mineral and organic constituents interact, may consequently also indirectly alter organic matrix ultrastructure and distribution. In the present work, the effects of demineralization have been investigated on the retention in chicken bone of two phosphoamino acids,O‐phosphoserine andO‐phosphothreonine, found in bone phosphoproteins proposed to be important in vertebrate mineralization and, more specifically, on the retention and distribution of a 66 kD bone phosphoprotein (66 kD BPP, osteopontin) also implicated in the calcification process. In tibiae fixed initially with 1% glutaraldehyde and then demineralized in 0.5 N HCl, 0.5 N acetic acid, or 0.1 M EDTA (all containing 1% glutaraldehyde), amino acid analyses and quantitative immunocytochemistry revealed that the phosphoamino acid content and the distribution of the 66 kD BPP were essentially the same as in fixed undemineralized controls. However, demineralization slightly altered the ultrastructural appearance of immunolabeled, electron‐dense patches of organic material in the bone matrix. In unfixed bone demineralized with any of these acids, there was a substantial loss of phosphoamino acids and the 66 kD BPP from the bone matrix. The relative ability of these acids to extract phosphoproteins from unfixed bone was found to decrease in the order EDTA>HCl>acetic acid. These results emphasize the differential effects on structural components of various demineralization and extraction procedures for biochemical and immunocytochemical studies of biologic tissues. Furthermore, they demonstrate that initial fixation with glutaraldehyde retains phosphoproteins in bone, with or without demineralization, while being adequate for immunocytochemical localization of certain bone matrix proteins and that an understanding of the action of specimen preparation on organic constituents (as well as inorganic components) is essential for accurately describing ultrastructural matrix‐mineral relati

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060907

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractRecently we reported that the osteoclast originates from the pluripotent hematopoietic stem cell. However, a detailed analysis of the progenitor and precursor stages of the osteoclast lineage is hard to perform with primary cultures of stem cells. In the present investigation interleukin‐3 (IL‐3) ‐dependent multipotent hematopoietic stem cells lines (FDCP‐mix), which have many characteristics in common with freshly isolated hematopoietic stem cells, were assayed for their osteoclast formation capacity. FDCP‐mix cell lines A4, C2GM, and 15S were cocultured with periosteum‐free 17‐day‐old fetal metatarsal bones. The effects of culture time, medium composition, and addition of WEHI‐3b‐conditioned medium (an unpurified IL‐3 preparation) on osteoclast formation were studied. 15S cells never differentiated into osteoclasts. Both A4 and C2GM cells were able to generate osteoclasts. Osteoclast formation was visualized by staining for tartrate‐resistant acid phosphatase activity and confirmed by45Ca release assays and electron microscopic studies. Medium supplemented with fetal calf serum clearly supported osteoclast formation from A4 cells better than medium supplemented with cock serum. The difference between fetal calf serum and horse serum is generally less pronounced. C2GM cells formed osteoclasts more readily and, generally, earlier than A4 under all culture conditions. WEHI‐3b‐conditioned medium addition increased the numbers of osteoclasts and their resorption activity. The coculture of stripped metatarsal bones with FDCP‐mix cell lines therefore offers a model system with many possibilities for the study of osteocla

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060908

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractThe effect of parathyroid hormone‐related protein (PTHrP) fragments 1–34, 38–64, and 67–86 on acetylcho‐line‐stimulated rat uterine contraction was examined in vitro. In addition, the possibility that PTHrP‐(38–64) or (67–86) influenced relaxation caused by PTHrP‐(1–34) was also investigated. Contraction of uterine horns was stimulated with 10−6or 10−5M acetylcholine. PTHrP‐(1–34) reduced the magnitude of acetylcho‐line‐stimulated uterine contraction. This effect was dose related over a concentration range of 10−9–10−6M. Neither PTHrP‐(38–64) or PTHrP‐(67–86) at concentrations of 10−8–10−6M affected uterine contraction stimulated by 10−6M acetylcholine. These fragments did not affect the relaxation caused by 10−7M PTHrP‐(1–34). These results demonstrate that (1) PTHrP‐(1–34) at 10−6M influences contraction of the rat myometrium and (2) the muscle relaxant activ

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060909

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractA single application of recombinant human transforming growth factor β1(rhTGF‐β1) adjacent to cartilage was found to induce bone formation in rabbit ear full‐thickness skin wounds. At doses that optimally promote soft tissue healing, 25–100 ng rhTGF‐β1per wound caused osseous tissue formation starting 21 days after wounding to reach a peak incidence and area of bone formation at day 42. Bone formation was followed by active remodeling, resulting in lower incidence and area of bone formation at days 56 and 70. The early phase of bone formation was located overlying the cartilage and involved perichondrial cells that appeared to differentiate directly into osteoblasts forming bone matrix without a cartilage precursor. Cartilage was replaced with bone at later time points. rhTGF‐β1was able to increase the ratio of osteoblasts to osteoclasts lining the trabecular surface of bone and thus increase the net amount of bone formation. The present studies suggest a potential therapeutic role for rhTGF‐β1in ha

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060910

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY

摘要:

AbstractBone cell populations obtained by sequential digestion of newborn mouse calvariae remain morphologically heterogeneous despite well‐documented biochemical differences. Fractionation of these populations on Percoll gradient reveal three major cell groups of low, intermediate, and high buoyant density (1.056, 1.070, and 1.095 g/ml) that are present in different ratios in early and late released populations. Cells of low and intermediate density dominate in early released populations. In contrast, late released populations contain mostly high‐density cells. Basal levels of alkaline phosphatase are highest in cells of intermediate buoyant density. All cells respond to PTH with cAMP production and morphologic transformation, but biochemical responses to PTH, such as secretion of insulin‐like growth factor I (IGF‐I) and stimulation of alkaline phosphatase activity, occur mostly in cells of intermediate density. These data suggest that (1) subclasses of osteoblasts can be further separated by density and (2) PTH effects on alkaline phosphatase activity and IGF‐I secretion are probably expressed by osteoblasts of a certain subclass and/or stage of de

ISSN:0884-0431    

DOI:10.1002/jbmr.5650060911

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1991

数据来源: WILEY