bluuebunny/crossref_metadata_2025_split huggingface dataset:
Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone
Authors: Dr. Hefley Thomas J. 
Published: February 2010

Abstract
Crude bacterial collagenase is essential for the enzymatic isolation of cells from the membranous bone of neonatal mouse calvaria. We have employed the newly developed methodology of fast protein liquid chromatography (FPLC) to separate and quantify the isozymes of collagenase so that their role in the enzymatic isolation of cells might be determined. FPLC resolved as many as six protein peaks in less than 30 min using a single anion exchange column and separated collagenase isozymes into two classes. The Class I isozymes had a preference for the substrate Azocoll, a denatured collagen substrate, and the Class II isozymes had a preference for Hexapeptide, a synthetic substrate. Two preparations of chromatographically purified collagenase (CGN-A and CGN-B) were tested for their ability to release viable cells from bone. Both preparations of purified collagenase completely digested the calvaria in 120 min. The total cell yield obtained with CGN-A was 0.34 million cells per calvarium. The yield obtained with CGN-B was 1.01 million cells per calvarium. Each preparation of purified collagenase was analyzed using FPLC. CGN-A contained only Class I collagenase isozymes, whereas CGN-B contained a mixture of both Class I and Class II isozymes. The collagenase isozymes of CGN-B were separated by FPLC and then combined in a 4:1 ratio of Class II:Class I isozymes. Utilization of FPLC-separated collagenase isozymes for the cell isolation increased the total cell yield to 1.50 million cells/calvarium. We have concluded that there are many combinations of collagenase isozymes that will completely digest the extracellular matrix of bone. However, only a combination which favors the Class II isozymes will result in a low rate of cell destruction and high cell yields.
Retinole acid and glucocorticoids enhance the effect of 1,25-dihydroxyvitamin D3 on bone γ-carboxyglutamic acid protein synthesis by rat osteosarcoma cells
Authors: Nishimoto Satoru K. , Salka Carl , Nimni Marcel E. 
Published: February 2010

Abstract
Two 1,25-dihydroxyvitamin D3-controlled parameters in the osteoblastlike osteosarcoma cell line ROS 17/2, bone γ-carboxyglutamic acid-containing protein (BGP) and collagen synthesis, were measured after pretreatments with either retinoic acid (RA), or triamcinolone acetate (TRM). RA and TRM both caused double the expected increase in BGP secretion at 16 hr after treatment with 1,25-dihydroxy vitamin D3. Triamcinolone acetate concentrations of 10−8 and 10−9M or 10−6M retinoic acid were effective in enhancing the 1,25-dihydroxyvitamin D3 stimulation of BGP secretion. Treatment with RA or TRM alone did not stimulate BGP secretion. RA alone had no effect on BGP secretion, while TRM inhibited BGP secretion. Collagen synthesis is inhibited by 1,25-dihydroxyvitamin D3. Neither retinoic acid nor triamcinolone acetate enhanced the 1,25-dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Retinoic acid by itself inhibited collagen synthesis but did not change the 1,25 dihydroxyvitamin D3-mediated inhibition of collagen synthesis. Triamcinolone acetate by itself or together with 1,25-dihydroxy vitamin D3 increased collagen synthesis.
We conclude that, although both triamcinolone acetate and retinoic acid increase the 1,25-dihydroxy vitamin D3 stimulation of BGP secretion by ROS 17/2 cells, they have different effects on the regulation of collagen production. Thus, although both hormones increase the 1,25-dihydroxy vitamin D3 receptor concentration in these cells, their actions are not mediated solely by this mechanism.
Ultracytochemical investigation of calcium-activated adenosine triphosphatase (Ca++-ATPase) in chick tibia
Authors: Akisaka Toshitaka , Yamamoto Toshio , Gay Carol V. 
Published: February 2010

Abstract
The ultrastructural distribution of Ca++-ATPase in bone cells of growing chick tibia was investigated by a cytochemical method in order to gain insight into possible sites of calcium ion translocation. Both osteoclasts and osteoblasts showed a polar distribution of reaction product along the plasma membrane. In osteoclasts, enzymatic activity occurred along the portion of the plasma membrane facing the marrow but not along the ruffled border or clear zone. The reaction product in these cells was due solely to Ca++-ATPase action. In osteoblasts, the plasma membrane facing away from bone (apical and lateral membrane) was very intensely stained, whereas the basal membrane was unstained. The reaction product in these cells appeared to be the result of both Ca++-ATPase and Ca++, Mg++-ATPase. In osteocytes, no plasma membrane staining was detectable. Mitochondrial staining in all three types of cells was more sensitive to fixation than was the plasma membrane enzyme, suggesting that mitochondrial and plasma membrane Ca++-ATPases are chemically distinct, as biochemical studies have shown. In general, mitochondria in osteoclasts stained more intensely than those in osteoblasts or osteocytes. Mitochondrial and vesicular sites of activity may be related to intracellular calcium storage, whereas calcium ATPases of the plasma membrane are presumed to be involved in calcium efflux from the cells. Calcitonin treatment did not alter the enzymatic distribution or intensity in osteoclasts. The striking polar distribution of both osteoclast and osteoblast plasma-membrane activity suggests that directional calcium pumping by these cells may be of importance in bone-forming and bone-resorbing mechanisms.
Regulation of alkaline phosphatase expression in a neonatal rat clonal calvarial cell strain by retinoic acid
Authors: Ng Kong Wah , Gummer Paul R. , Michelangeli Valdo P. , Bateman John F. , Mascara Thomas , Cole William G. , Martin T. John 
Published: February 2010

Abstract
A clonal cell strain, UMR 201, was established from a culture of rat calvarial cells by the process of limiting dilution on a collagen substratum. One-day-old neonatal rat calvaria stripped of periosteum were placed on collagen in α-MEM with 10% fetal bovine serum (FBS). Cells that grew out from the calvaria were passaged eight times to select cells with the ability to proliferate in culture before cloning was attempted. Cells from the clonal strain were homogeneous in appearance with a doubling time in culture of about 24 hours. The UMR 201 cells formed predominantly type 1 collagen. When treated with retinoic acid (RA), all cells showed an intense staining for alkaline phosphatase (ALP). This effect of RA on the expression of ALP activity was reversible and was time and dose dependent. The earliest change was observed within 6 hours. In contrast, single and isolated clumps of untreated cells stained positively for ALP only when they were confluent. Coincubation with dactinomycin up to 3 hours after the addition of RA completely prevented the expression of ALP, whereas dactinomycin became progressively less effective when added at later times. This is interpreted as indicating a regulatory role of RA on the gene expression of ALP. Other hormones acting on bone, such as 1,25(OH)2 vitamin D3 and dexamethasone, also modulate ALP activity. The cells showed morphologic evidence of senescence after passage 12. Our preliminary studies showed that the UMR 201 cells had the characteristics of relatively undifferentiated mesenchymal cells. They will be useful models to study the regulation of ALP activity and type 1 collagen synthesis by various factors and the induction of expression of other phenotypic characteristics by RA.
Phorbol esters stimulate bone resorption in fetal rat long-bone cultures by mechanisms independent of prostaglandin synthesis
Authors: Lorenzo Joseph A. , Sousa Sandra 
Published: February 2010

Abstract
The phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate, which activate the enzyme protein kinase C, stimulated resorption in fetal rat long-bone cultures at concentrations of 1 and 10 μM. This effect appeared specific for active phorbol esters, since the inactive analogue 4-alpha-phorbol-12,13-didecanoate was without effect. The resorptive responses of fetal rat long-bone cultures to active phorbol esters differed from those previously described in newborn mouse calvaria cultures, since resorption stimulated by TPA in the rat long bones was not inhibited by either indomethacin (10 μM) or flufenamic acid (10 μM). However, calcitonin, an inhibitor of osteoclastic resorption, did decrease the response to TPA. There were some similarities between the response of fetal rat long-bone cultures to TPA and their response to epidermal growth factor (EGF). Like EGF, TPA stimulated DNA synthesis in the bones (measured as the incorporation of [3H]-thymidine) at concentrations below those necessary to stimulate resorption. TPA also did not stimulate resorption in the presence of aphidicolin (10 μM), an inhibitor of DNA synthesis that has been previously shown to block the resorptive response of these cultures to EGF. However, the responses of the cultures to TPA and EGF were not identical, since, unlike the effects of EGF, the stimulatory effects of TPA on DNA synthesis were biphasic. These results demonstrate that active phorbol esters stimulate bone resorption in fetal rat long-bone cultures through mechanisms that do not require prostaglandin synthesis but do appear to be mediated by osteoclasts. Hence, it is possible that one mechanism by which hormones stimulate resorption in bone is through activation of protein kinase C.
Growth and repair of cartilage: Organ culture system utilizing chondroprogenitor cells of condylar cartilage in newborn mice
Authors: Weiss Anna , Livne Erella , von der Mark Klaus , Heinegard Dick , Silbermann Michael 
Published: February 2010

Abstract
The zone of progenitor cells of mandibular condyles of neonatal mice was kept in an organ culture system for up to 8 days. Qualitative and quantitative determinations indicated a pronounced proliferative activity during the initial phases of the culture followed by a differentiation phase and the acquisition of typical hyaline cartilage. The mature hypertrophic chondrocytes were found to be surrounded by cartilage-specific macromolecules such as type II collagen, cartilage proteoglycans, and cartilage anchorin. The extracellular mineralization proceeded along matrix vesicles as is usually noted in vivo. A unique finding in this study was the observation that explants comprising cartilage progenitor cells and their adjacent extracellular matrix succeeded in repairing the damaged condylar in vitro.
Multinucleated cells elicited in response to implants of devitalized bone particles possess receptors for calcitonin
Authors: Goldring Steven R. , Roelke Merrilee , Glowacki Julie 
Published: February 2010

Abstract
The introduction into soft tissues of particulate materials resistant to digestion results in the induction of a “foreign-body giant-cell reaction.” We have examined the relation between osteoclasts and foreign-body giant cells by comparing the tissue responses elicited by subcutaneous implants of devitalized, mineral-containing bone particles (BP), nonresorbable plastics such as polymethylmethacrylate (PMMA), or both. Implantation of BP results in the recruitment of multinucleated cells with features of in osso osteoclasts including tartrate-resistant acid phosphatase activity, contact-mediated resorption of BP, membrane specializations (ruffled borders and clear zones), and inhibition of resorption by calcitonin treatment of animals. In the present study, an autoradiographic technique employing 125I-salmon calcitonin was used to demonstrate the presence of receptors for this hormone on multinucleated cells from BP implants. In contrast, outgrowth cells from PMMA implants lacked calcitonin receptors. Demonstration of features of the osteoclastic phenotype in multinucleated cells elicited in response to BP supports the hypothesis that the mineralized matrix of bone may be a requirement for acquisition of the osteoclast phenotype.
Static and tetracycline-based bone histomorphometric data from 34 normal postmenopausal females
Authors: Dr. Recker Robert R. , Kimmel Donald B. , Parfitt Michael A. , Davies Michael K. , Keshawarz Naser , Hinders Sharilyn 
Published: February 2010

Abstract
Transilial bone biopsies were obtained from 34 healthy postmenopausal women following in vivo fluorochrome labeling. Stained and unstained undecalcified sections were evaluated using a Merz grid. Standard histomorphometric data from cancellous bone tissue were collected and the results were evaluated and presented as variables commonly used in bone histomorphometry. The normal ranges, medians, means, and standard deviations for the group of 34 are presented in tabular form for structural, surface, basic dynamic, and derived dynamic data. Similar data for individuals grouped by ages 45–54, 55–64, and 65–74 are also presented. Secular trends for the whole group are evaluated. The structural and surface data are not much different from previous reports of sudden-death accident victims, when methodologic differences are considered. The mineral apposition rate (MAR) was 0.53 ± 0.08 μm/day, similar to previous reports in cancellous bone, but one-third less than in cortical bone. MAR showed a marked decline with age. In contrast, the extent of tetracycline-labeled surfaces varied widely without a secular trend. Double-label surface (dLS/BS) ranged from 0.5 to 8.0% and single-label surface (sLS/BS), from 0.5 to 10.5%. Mineralizing osteoid surface (MS/OS) varied from 2 to 64%. Using only double-label surface to represent mineralizing surface, volumebased bone formation rate (BFR/BV) ranged from 0.7 to 28%/yr, and the remodeling period (Rm.P) varied from 0.28 to 4.5 years. Calculations using other representations of mineralizing surface [double plus one-half single label (MS/BS″); all label (MS/BS′)] are also presented. These bone histomorphometric data are important because: (1) they come from a cohort of living subjects that was recruited solely for the purpose of establishing normal bone histomorphometry; (2) they represent the age range of patients with postmenopausal osteoporosis; and (3) they markedly expand the bone histomorphometric database of healthy persons given in vivo fluorochrome labeling prior to transilial biopsy.
Interaction of calcium nutrition and physical activity on bone mass in young women
Authors: Kanders Bonnie , Dempster David W. , Lindsay Robert 
Published: February 2010

Abstract
We have examined the relationships among calcium intake, mechanical stress (activity) and axial (lumbar) and peripheral (midradius) bone mass in a population of 60 young normal, healthy eumenorrheic women (age 25–34 years), selected on the basis of weight for height (within 20% of expected). In this population, vertebral bone mineral density was significantly related (r = 0.41, p &lt; 0.005) to activity pattern but not to calcium intake. Elimination of the effects of activity, however, disclosed a significant correlation between total calcium intake and vertebral bone mass (r = 0.36, p &lt; 0.02). Closer examination of this relationship suggested that vertebral bone mineral density did not appear to increase with calcium intakes above 800–1000 mg/day, implying a threshold effect and vertebral density was better expressed as a function of the logarithm of calcium intake (r = p &lt; 0.01). Radial mineral content was not related to activity (which was mostly walking and running), but it was significantly affected by calcium intake. These data suggest that skeletal status in the vertebrae in young women may be influenced by modulation of mechanical stress. However, bone mineral density is probably also dependent on nutritional status such that the optimum calcium intake for this effect is 800–1000 mg/day. If such effects, observed here cross-sectionally, can be substantiated in longitudinal studies and persist into the aging postmenopausal population, then the average age of onset of osteoporotic fractures would be expected to be delayed approximately 10 years, by relatively simple modifications of diet and activity.
Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate
Authors: Dr. Feyen Jean H.M. , Petersen Donna N. , Kream Barbara E. 
Published: February 2010

Abstract
We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3–100 ng/ml (4–133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.
Forskolin augments the effects of calcitonin on cytoplasmic spreading of isolated rat osteoclasts and plasma calcium levels in the rat
Authors: Nicholson G.C. , Yumita S. , Moseley J.M. , Yates A.J.P. , Martin T.J. 
Published: February 2010

AbstractRecently we have shown that forskolin acts synergistically with calcitonin on stimulation of cyclic AMP (cAMP) production in isolated rat osteoclasts and now show that forskolin also augments later physiological responses to calcitonin. The sensitivity of calcitonin inhibition of cytoplasmic spreading in isolated rat osteoclasts was increased 3.5-fold by simultaneous treatment with 10−7M forskolin, which alone had no effect on the response. In weanling rats the threshold of the hypocalcemic response to salmon calcitonin was reduced fivefold by simultaneous treatment with 200 μg/kg forskolin i.v., which alone did not influence the plasma calcium. These results provide further evidence that calcitonin-induced inhibition of osteoclast function is mediated by elevation of cAMP levels and suggest that forskolin could be used to augment the therapeutic effects of calcitonin.
Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: The effect of a bisphosphonate
Authors: Dr. Löwik Clemens W.G.M. , Van Der Pluijm G. , Van Der Wee-Pals L.J.A. , Van Treslong-De Groot H. Bloys , Bijvoet O.L.M. 
Published: February 2010

Abstract
Osteoclast-devoid bone explants were cultured together with embryonic liver as a source of osteoclast precursors, but separated from each other by a filter. Cells migrated through the filter toward the calcified matrix and acquired the characteristics of mature, tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts upon contact with the bone explant. Migration and attachment could be visualized separately. Progressive reduction of filter pore size resulted in progressive reduction of resorption because the use of smaller pores made it increasingly difficult for cells to pass. Indeed, the use of 0.22-micron filters, through which no cells can pass, but which still allow full passage of medium, completely blocked the resorption. When migrating cells from fetal liver were arrested for 10 days by using a combination of filters with different pore sizes, the arrested cells showed a tendency to fuse just opposite the mineralized matrix. Furthermore, a great number of the arrested cells expressed the macrophage-specific cell-surface antigen F4/80 and showed acid phosphatase activity, but none of these cells were tartrate resistant. The acquisition of tartrate-resistant acid phosphatase activity upon contact with the bone explant and subsequent resorption of this explant could be prevented by exposure of the system to the bisphosphonate dimethyl-APD (Me2-APD), whereas migration of cells through the filter was not affected. We suggest that the bisphosphonate interferes with a matrix factor that is essential for the attachment and subsequent transformation of the osteoclast precursor into the mature phenotype.
Dose dependency of calcium absorption: A comparison of calcium carbonate and calcium citrate
Authors: Harvey Jean A. , Zobitz Margaret M. , Pak Charles Y.C. 
Published: February 2010

Abstract
Calcium supplementation is recommended as a prophylaxis against bone loss. This study was performed to determine the dose dependency of calcium absorption in an attempt to derive an optimum dose schedule. Using the well-described oral calcium load technique, we measured the calcium absorption from three different calcium doses (0.5, 1.0, and 2.0 g) of both calcium carbonate and calcium citrate administered to 21 normal subjects (4 men and 17 women, 22–60 years). Nine subjects underwent two additional loads with 0.2 g of elemental calcium as calcium carbonate and as calcium citrate. The intestinal calcium absorption from calcium carbonate and calcium citrate was estimated from the rise in urinary calcium following oral ingestion of the respective calcium salt. The increment in urinary calcium post-load, reflective of intestinal calcium absorption, rose rapidly from 0 to 0.5 g calcium loads with only slight subsequent increases from the 0.5 g to 2.0 g calcium doses. Thus, results indicate that 0.5 g of calcium is the optimum dose of either calcium salt. Moreover, the increment in urinary calcium post-load was higher from calcium citrate than from calcium carbonate at all four dosage levels. The increment in urinary calcium (during the second 2 hr) following calcium citrate load (0.5 g calcium) was 0.104 ± 0.096 mg/dl glomerular filtrate (GF), which was higher than that of 0.091 ± 0.068 mg/dl GF obtained from 2.0 g calcium as calcium carbonate. These results confirm the superior calcium bioavailability from calcium citrate as compared with calcium carbonate. To confirm findings from the calcium load technique, 9 subjects underwent calcium absorption test from the fecal recovery of orally administered radiolabeled calcium salts (containing 100–200 mg of elemental calcium). Fractional calcium absorption from calcium citrate of 0.402 ± 0.067 was significantly higher than that of 0.314 ± 0.100 obtained from calcium carbonate. Moreover, calcium loading with either salt did not produce significant citraturic effect during 4 hr of study, ruling out “calcium trapping” by citrate in the renal tubule. In conclusion: (1) Both calcium carbonate and calcium citrate display dose dependency of calcium absorption; (2) calcium is more absorbable from calcium citrate than from calcium carbonate at all four doses employed (0.2, 0.5, 1.0, and 2.0 g); and (3) calcium absorption from 0.5 g of calcium as calcium citrate is greater than that from 2.0 g of calcium as calcium carbonate.