摘要:

AbstractA model was developed for the application of cyclic mechanical loads to 17 day embryonic chick tibiotarsi in culture. A single 20 minute period of intermittent loading at 0.4 Hz, producing physiologic peak strains and strain rates, resulted in two peak strain magnitude‐related responses that were previously reported in vivo: (1) a rapid increase in glucose 6‐phosphate dehydrogenase activity in osteoblasts and osteocytes and (2) increased RNA synthesis, as shown by increased incorporation of [3H]uridine into extracted RNA. The RNA response was detectable 8 h following loading but was more pronounced by 24 h. Both responses were blocked by indomethacin (10−6M). These results demonstrate that embryonic chick bones in organ culture exhibit cellular responses to loading similar to those previously identified in adult canine cancellous bone cultures in vitro and adult avian cortical bone in vivo. These findings are consistent with a sequence of events between loading and new bone formation that includes an immediate strain magnitude‐related, prostanoid‐dependent increase in activity of the pentose monophosphate shunt in osteoblasts and osteocytes, followed by a similarly strain magnitude‐related increase in RNA synthesis over the subs

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080302

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe tibiae and fibulae of 14‐week‐old rats were subjected to a single 5 minutes period of cyclic longitudinal loading at 1 Hz. The activity of the enzymes glucose 6‐phosphate dehydrogenase (G6PD) and alkaline phosphatase (ALP) in osteocytes and periosteal osteoblasts was measured immediately and 24 h after loading. In osteocytes G6PD activity was increased immediately after loading but returned to control values 24 h later. There was no detectable ALP activity in these cells regardless of loading history. In periosteal osteoblasts G6PD activity was raised immediately after loading and remained higher than controls 24 h later. ALP activity in periosteal cells was unaffected immediately after loading but 24 h later was substantially increased. These findings are consistent with osteocytes and periosteal cells both being immediately responsive to periods of intermittent loading in their adjacent matrices. In both cell types an early feature of this response is an increase in G6PD activity. In osteocytes this response is short‐lived, suggesting that it is an early biochemical change associated with strain perception that does not progress to matrix synthesis. The increase in G6PD activity with unaffected ALP levels in periosteal cells immediately after loading is consistent with a similar response. In these cells the increase in G6PD accompanied by increased ALP levels 24 h after loading suggests that the loading‐related response progresses to new bone

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080303

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

年代:1993

数据来源: WILEY

摘要:

AbstractHypercalcemia in human granuloma‐forming diseases like sarcoidosis results from the endogenous overproduction of 1,25‐dihydroxyvitamin D [1,25‐(OH)2D] by disease‐activated tissue macrophages. The recent identification of an immortalized chick myelomonocytic cell line, HD‐11, that constitutively expresses a 25‐hydroxyvitamin D (25‐OHD) 1‐hydroxylation reaction has alleviated dependence on studying primary macrophage cultures with no replicative potential in vitro. In these experiments we established conditions for the maximal expression of the HD‐11 cell 25‐OHD3‐1‐hydroxylation reaction and localized this activity to the mitochondrial fraction. On a per cell basis, the activity of HD‐11 cell 25‐OHD31‐hydroxylation reaction was comparable to that in primary cultures of chick renal tubular epithelial cells, which express the authentic renal 25‐OHD31‐hydroxylase. Maximal product yield was achieved after incubation of HD‐11 cells with 200 nM 25‐OHD3for 3 h. Although adherent monolayers possessed 3‐ to 4‐fold more capacity for hormone production than cells in suspension, suspended cells exhibited easily detectable 25‐OHD3catalytic activity (0.58 ± 0.08 pmol per 106cells per h; ± SEM), 50% of which remained solubilized in a sonicate of suspended cells cleared of nuclei and plasma membrane. Subcellular localization disclosed 91% of the residual activity to be concentrated in the mitochondrial subfraction. A detergent‐solubilized extract of this mitochondrial subfraction contained 1.9 ± 0.3 pmol 1,25‐(OH)2D3synthetic capacity per mg protein. The catalytic activity (1‐hydroxylase activity) was concentrated 20.2‐fold after chromatography on octyl‐amino agarose and was associated with 0.054 nmol cytochrome P450per mg protein. These data suggest that HD‐11 cell 25‐OHD31‐hydroxylation reaction may be similar to the w

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080304

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

年代:1993

数据来源: WILEY

摘要:

AbstractTwo sets of clonal cell populations differing in the expression of osteoblastic traits, the rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 and the immortalized fetal rat calvarial cell lines RCT‐1 and RCT‐3, were compared for their ability to attach to a series of extracellular matrix (ECM) constituents in vitro. Both osteoblastic (ROS 17/2.8, RCT‐3) and nonosteoblastic (ROS 25/1, RCT‐1) cell lines attached in a time‐ and concentration‐dependent manner to plates coated with fibronectin (FN), osteopontin (OP), type I collagen (Col I), type IV collagen (Col IV), and laminin (LN) but only weakly to osteocalcin (OC) and thrombospondin (TSP). In both systems, the osteoblastic and nonosteoblastic clones attached identically to FN. Both ROS 17/2.8 and ROS 25/1 attached to similar molar amounts of substrate with the same preference order: FN>LN>Col I ≥ Col IV. Maximal ROS 17/2.8 attachment to OP was ≥ Col I but required approximately 2.5 times more substrate. ROS 25/1 attached less effectively than ROS 17/2.8 to most non‐FN substrates. RCT‐3 cells attached similarly to ROS 17/2.8 except that the preference order for Col I and LN was reversed and attachment to OP was lower than for ROS 17/2.8 RCT‐1 cells attached best to Col I rather than FN, and equaled or surpassed RCT‐3 in attachment to other non‐FN substrates. Thus in these experimental systems, cells expressing an osteoblast‐like phenotype exhibited generally similar ECM attachment properties. Their nonosteoblastic counterparts recognized the same spectrum of ECM constituents but differed from the osteoblastic cells and from each other in the effectiveness of their attachment

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080305

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe established cultures of cells growing out from adult bone chips and maintained them through 12 passages in culture. The cultures showed osteoblastic phenotype accompanied by synthesis of collagen type I, osteonectin, alkaline phosphatase, and osteocalcin. We report the chracterization of 21 clones obtained from three different individual primary cultures. We studied the expression of osteonectin, alkaline phosphatase, collagen, and osteocalcin in the clones. Metabolic labeling showed production of type I collagen and of osteonectin in all clones studied. In two‐thirds of the clones and in mass cultures alkaline phosphatase was not detected at passage 2, but it was detected in increasing amounts at later passages in culture. The clones attained different but detectable levels of expression of this marker by passage 8. The different levels in the expression of alkaline phosphatase in positive clones may be because they were derived from cells at different stages of osteoblastic maturation or due to small changes in microenvironment. The alkaline phosphatase‐positive clones were tested for osteocalcin, and they showed measurable expression only at passage 10. A third of the clones obtained were negative for alkaline phosphatase during 12 passages in culture. The obtainment of clones unable to produce alkaline phosphatase may be due to loss of differentiating potential under the in vitro culture conditions. The growth rate and potential of all clones studied were similar through 12 passages in culture, regardless of their potential for expression of alkaline phosphat

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080306

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe intrinsic (material) quality of cancellous and cortical bone was evaluated in vivo from the measurement of reflection ultrasound velocities in the ulna. In cancellous bone, the reflection ultrasound velocity was inversely correlated with age in normal women (r= −0.48,p= 0.001), with a significantly lower mean value in 32 normal postmenopausal women than in 14 premenopausal women (3124 versus 3341 m/s,p<0.0001). In 32 untreated osteoporotic women the cancellous bone velocity was lower than in normal postmenopausal subjects (2906 versus 3124 m/s,p= 0.0001). Following treatment with slow‐release sodium fluoride plus calcium citrate (mean 2.4 years in 33 osteoporotic patients with no fracture during treatment), the cancellous bone velocity was significantly higher than in untreated osteoporotic women (3082 versus 2906 m/s,p= 0.0002) and was not significantly different from that in normal postmenopausal women. The cortical bone velocity displayed similar trends, but the changes did not attain statistical significance. The measurements were repeated approximately 9 months later in 9 untreated and in 20 treated patients; in 5 additional patients, the measurements were made both before and after 9 months of treatment with slow‐release sodium fluoride and calcium citrate. The cancellous bone velocity increased significantly (p= 0.046) in these patients, from 3008 m/s before treatment to 3112 m/s after the first 9 months of treatment. The velocity rose significantly from 3037 to 3167 m/s (p= 0.017) in patients treated for a short time (12–30 months at first measurement), but it did not change in untreated patients or those treated for more than 30 months. Thus, the material quality of cancellous bone decreases with normal aging and is reduced further with the osteoporotic process. This impaired quality may be corrected by treatment with slow‐release sodium fluoride plus calciu

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080307

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

年代:1993

数据来源: WILEY

摘要:

AbstractA method for sensitive quantitation of bone gla protein (BGP, osteocalcin) mRNA has been developed using competitive polymerase chain reaction after reverse transcription (competitive RT‐PCR). The complementary DNA (cDNA) reverse transcribed from sample RNA was coamplified in a PCR with a known amount of mutant BGP cDNA (competitor) using the identical oligonucleotide primers. The mutant cDNA with its unique restriction site allowed quantitation of sample and mutant PCR products after densitometric analysis of ethidium bromide‐stained agarose gels. A linear relationship between initial sample BGP amount and the ratio of BGP to mutant BGP band intensity was obtained and used to make a standard curve to determine the initial BGP mRNA of unknown samples. These standard curves were made with known amounts of recombinant BGP cDNA. The competitive RT‐PCR for BGP allows measurement of twofold differences in 1 and 2 μg total RNA and requires at least 10 times less sample RNA than usual Northern blotting. Moreover, heteroduplexes with one BGP strand and one mutant BGP strand formed as a result of high PCR cycles were quantifiable. This provided the advantages of rapid quantitation from ethidium bromide‐stained gels without blotting, hybridization, or autoradiography. Multiple samples could be assayed for greater confidence in the results. The sensitivity, accuracy, and ease of the assay will facilitate analysis of BGP mRNA from a small amount of sample. The assay has been used to confirm the BGP mRNA changes with hormonal treatment in cultured cells and the age‐related changes in whole tib

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080308

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

年代:1993

数据来源: WILEY

摘要:

AbstractThere has been recent evidence that calcium/protein kinase C (Ca/PKC) messenger system as well as adenylate cyclase are involved in the signal transduction stimulated by PTH. We therefore examined the role of these dual‐signal transduction systems and the interaction of these systems in the regulation of DNA synthesis by PTH in the osteoblastic osteosarcoma cells, UMR‐106. As recently reported, 10−4M Sp‐cAMPS, a direct activator of cAMP‐dependent protein kinase (PKA), and 10−4M dibutyryl‐cAMP, as well as hPTH‐(1–34), caused the significant inhibition of [3H]thymidine incorporation (TdR). Both A23187 and ionomycin (10−8‐10−6M) inhibited TdR in a dose‐dependent manner, with a minimal effective dose at 10−7M. Although 10−6M phorbol 12‐myristate 13‐acetate (PMA) caused slight but significant stimulation of TdR by itself, it augmented not only dibutyryl‐cAMP‐ but also Sp‐cAMPS‐induced inhibition of TdR. On the other hand, 4α‐phorbol 12,13‐didecanoate, incapable of activating PKC, failed to augment these cAMP analogs‐induced effects. Pretreatment with 50 μM H‐7, an inhibitor of PKC, not only abolished the PMA‐induced augmentation of effect by cAMP analogs but also significantly blocked the PTH‐induced inhibitory effect on TdR. Pretreatment with 10−6M PMA, which downregulates PKC, significantly inhibited the PTH‐induced suppression of TdR. Combined treatment with cAMP analog (dibutyryl‐cAMP or Sp‐cAMPS) and calcium ionophore (A23187 or ionomycin) caused additive effects on TdR, and PMA used in combination with both cAMP analog and calcium ionophore induced the further inhibition of TdR. The present study indicated that in addition to the PKA system, the Ca/PKC system also played a contributory role in the full expression of PTH response in the regulation of DNA synthesis in osteoblasts. The present data provided additional evidence for a dual pathway of target cell activation by PTH and the existence of interac

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080309

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe investigated the effect of medium pH on activity of isolated osteoclasts and have also looked at the possibility that medium pH affects osteoclast numbers during culture. Osteoclast‐containing cell suspensions prepared from neonatal rabbits were cultured on bovine bone slices at pH 6.5, 7.0, or 7.5. After 24 or 48 h of culture, the cells and bone slices were fixed and stained for tartrate‐resistant acid phosphatase (TRAP). After counting the osteoclasts, the cells were removed and the resorption lacunae stained by immunostaining using anticollagen type I antibody and then quantitated. We found that the resorptive activity of isolated rabbit osteoclasts was sharply increased at pH 6.5–7. Osteoclast differentiation and proliferation, on the other hand, were optimal at pH 7.0–7.5 but decreased at pH 6.5. The results thus imply that pH regulation of the bone surface environment can dramatically alter both the number of osteoclasts and their resorptive a

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080310

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

年代:1993

数据来源: WILEY

摘要:

AbstractBurn patients are at risk for bone disease due to aluminum (AI) exposure from use of antacids and albumin, partial immobilization, and increased production of endogenous glucocorticoids. Moreover, severely burned children are growth impaired up to 3 years after the burn. To determine the extent of bone disease, we studied nine men and three women, ages 18–41 years, with greater than 50% body surface area burn. Seven patients underwent iliac crest bone biopsy following double tetracycline labeling, one additional patient expired after a single label, and three others had postmortem specimens obtained for quantitative AI only. Serial serum and urine samples were obtained weekly until biopsy or death. All biopsied patients had reduced bone formation and osteoid area, surface, and width, with mineral apposition rate, osteoblast surface, and osteoclast number with normal eroded surfaces compared to age‐ and sex‐matched normal ambulatory volunteers. Burn patients also had reduced bone formation, mineral apposition rate, osteoid area, and surface compared to age‐matched volunteers at short‐term bed rest. Serum levels of osteocalcin were low. Most patients had mild hypercalcemia but only a third had hypercalciuria. All patients had elevated AI in blood or urine; urine AI correlated inversely with serum osteocalcin. In 60% significant bone AI was detectable by stain or quantitation. Our data are compatible with burn patients having markedly reduced bone turnover. AI loading, partial immobilization, endogenous corticosteroids, and cytokine production may be among the etiologi

ISSN:0884-0431    

DOI:10.1002/jbmr.5650080311

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

年代:1993

数据来源: WILEY