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O Ljunggren

Publications and source records attributed to O Ljunggren.

At least 37 records · Page 2Linked to original sources
PubMed1999

Three isolation techniques for primary culture of human osteoblast-like cells: a comparison.

The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOBcol). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihydroxyvitaminD3 (1,25(OH)2D3)-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)2D3-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexamethasone in the presence of ascorbic acid (50 microg/mL) and beta-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.

Adolescent↗
PubMed1999

Thrombin, but not bradykinin, stimulates proliferation in isolated human osteoblasts, via a mechanism not dependent on endogenous prostaglandin formation.

Osteolysis or osteosclerosis often occurs in bone tissue adjacent to chronic inflammatory processes. Numerous cytokines and inflammatory mediators have been implicated as osteoclast-activating agents, explaining inflammation-induced bone resorption. In many cases, the cause of the sclerosis seen in these lesions is less thoroughly investigated. We have studied the effects of thrombin and bradykinin, 2 inflammatory mediators, on the rate of proliferation in isolated human osteoblasts (hOBs). Thrombin, at and above 1 U/mL, stimulated the rate of thymidine incorporation into hOBs. The absolute cell number also increased, as measured by an assay based on the detection of cell metabolism. A synthetic peptide ligand for the thrombin receptor enhanced the rate of [3H]thymidine incorporation in hOBs, indicating that thrombin-induced proliferation is mediated via the tetheric thrombin receptor. The thrombin-induced proliferation was not affected by indomethacin, excluding prostanoids as mediators of this effect. Bradykinin did not affect either the rate of thymidine incorporation, or number of cells in long-term cultures of hOBs. In conclusion, the inflammatory mediator, thrombin, stimulates proliferation in isolated human osteoblasts probably via the recently described G-protein-coupled tetheric thrombin receptor. Thrombin may therefore be involved as a mediator of inflammation-induced sclerosis and bone formation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

Tumor necrosis factor-alpha and -beta upregulate the levels of osteoprotegerin mRNA in human osteosarcoma MG-63 cells.

Osteoprotegerin (OPG) is a recently cloned soluble member of the tumor necrosis factor receptor family. OPG has been shown to inhibit osteoclast recruitment by binding to OPG-ligand, an osteoclast differentiating factor on osteoblastic stromal cells, thereby blocking osteoclastogenesis. In this report we have examined the effect of tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) on OPG mRNA levels in the human osteosarcoma cell line MG-63. We demonstrate that both TNF-alpha and TNF-beta dose- and time-dependently upregulate the mRNA levels of OPG. The effect is significant at and above 5 pM of TNF-alpha and 1 pM of TNF-beta. The stimulatory effect on OPG mRNA levels in MG-63 cells was detected after 2 hrs of incubation with TNF-alpha or TNF-beta. These data demonstrate that the expression of OPG in osteoblasts, with subsequent effects on osteoclastogenesis, is regulated by TNFs.

Bone Neoplasms↗
PubMed1998

Osteoprotegerin mRNA is increased by interleukin-1 alpha in the human osteosarcoma cell line MG-63 and in human osteoblast-like cells.

Osteoprotegerin (OPG) is a soluble receptor for the Osteoprotegerin-Ligand (OPGL) which is expressed on osteoblasts and mediates the signal for osteoclast differentiation. In the present study we demonstrate that OPG mRNA levels in MG-63 cells are increased in a dose-dependent manner after 8 h of treatment with IL-1 alpha (338 +/- 53% over control at 25 U/ml). Interleukin-6 (IL-6), under similar culture conditions, does not affect OPG mRNA levels. Time-course studies show that IL-1 alpha (25 U/ml) causes a transient increase of OPG mRNA levels in MG-63 cells, peaking after 4 h of treatment. An increase of the OPG transcript occurs in hOB cells at 0.5 h which is still present after 24 h of IL-1 alpha treatment. In MG-63 cells neither basal-nor IL-1 alpha-induced OPG mRNA levels are altered by the translational inhibitor cycloheximide. These results suggest that expression of OPG in osteoblasts may be regulated by IL-1 alpha.

Bone Neoplasms↗
PubMed1998

Regulation of osteoprotegerin mRNA levels by prostaglandin E2 in human bone marrow stroma cells.

The recently cloned osteoclastogenesis inhibitory factor, or osteoprotegerin (OPG), has been shown to be a potent inhibitor of osteoclast formation. The inhibition is believed to be mediated through specific binding of OPG to a cell surface ligand on osteoblastic stromal cells. In this report we have studied the effect of the bone resorbing agent prostaglandin E2 (PGE2) on OPG mRNA levels in primary cultures of human bone marrow stroma cells (hBMSC). PGE2 dose- and time-dependently down-regulated the mRNA levels of OPG, as measured by RNAse protection assay. After 4 hours of stimulation with 1 microM PGE2, OPG mRNA levels were significantly decreased. The inhibitory effect was seen at and above 1 nM of PGE2. To elucidate whether the OPG mRNA levels are regulated via the proteinkinase A and/or the proteinkinase C pathways we stimulated cells with either forskolin (FSK) or phorbolic ester (PDbu) respectively. FSK (10 microM) decreased OPG mRNA levels to 50 % of control, whereas PE (10 nM) upregulated the mRNA levels to 250 % of control. These data show that PGE2 down-regulates the expression of OPG mRNA in hBMSC, probably via an increase in cAMP. This mechanism might be involved in PGE2-induced bone resorption.

Base Sequence↗
PubMed1998

Bone resorption induced by A23187 is abolished by indomethacin: implications for second messenger utilised by parathyroid hormone.

Parathyroid hormone acts on the osteoblast to induce osteoclastic bone resorption. Parathyroid hormone utilises cyclic AMP as a second messenger in osteoblasts, but may also cause an increase in cytoplasmatic free calcium ions ([Ca2+]i) in the same cell. To investigate the role of osteoblastic [Ca2+]i in the induction of bone resorption, we have compared the effects of parathyroid hormone and the Ca2+-ionophore, A23187, as well as the adenylate cyclase stimulating agent, forskolin, and the phorbol ester, phorbole 12,13 dibutyrate (PDB), on bone resorption in neonatal mouse calvarial bones. Parathyroid hormone (0.1 and 1 nM) dose dependently stimulated the release of prelabelled 45Ca2+ in 72 h culture. Parathyroid hormone-induced bone resorption was not affected by the addition of 1 microM indomethacin to the incubation media, and was therefore, not mediated by local prostaglandin formation. A23187 stimulated the release of 45Ca2+ at 1-10 nM. Above 100 nM, A23187 inhibited bone resorption. The A23187 (3 and 10 nM)-induced bone resorption was abolished by the cyclooxygenase inhibitor, indomethacin (1 microM), indicating that the stimulatory effect was mediated via prostaglandin formation. The adenylate cyclase stimulating agent, forskolin, dose dependently stimulated bone resorption at and above 1 microM. There was no additive or synergistic effect of forskolin and A23187 on 45Ca2+ release. Forskolin-induced bone resorption was, as with parathyroid hormone but in contrast to ionophore-induced bone resorption, not abolished by indomethacin (1 microM). The protein kinase C activator, PDB, at 10 and 1000 nM stimulated the release of prelabelled 45Ca2+. The stimulatory effect of the protein kinase C stimulating phorbol ester, PDB, on bone resorption was abolished by the addition of indomethacin. In summary, bone resorption induced by a Ca2+-ionophore is abolished by indomethacin. This indicates that bone resorbing agents known to increase [Ca2+]i subsequently enhance local prostaglandin formation. Bone resorption induced by the protein kinase C activator, PDB, was also abolished by indomethacin, whereas, forskolin and parathyroid hormone-induced bone resorption was unaffected. These data indicate that cyclic AMP, but not [Ca2+]i, is involved as a second messenger in parathyroid-induced bone resorption.

Animals↗
PubMed1998

Skeletal metastases from breast cancer: uptake of 18F-fluoride measured with positron emission tomography in correlation with CT.

OBJECTIVE: To characterise the uptake of 18F in skeletal metastases from breast cancer using positron emission tomography (PET) and to relate these findings to the appearance on CT. PATIENTS AND DESIGN: PET with 18F and CT were performed in five patients with multiple skeletal metastases from breast cancer. The CT characteristics were analysed in areas with high uptake on the PET study. Dynamic PET imaging of the skeletal kinetics of the 18F-fluoride ion were included. RESULTS: The areas of abnormal high accumulation of 18F correlated well with the pathological appearance on CT. Lytic as well as sclerotic lesions had markedly higher uptake than normal bone, with a 5-10 times higher transport rate constant for trapping of the tracer in the metastatic lesions than in normal bone. CONCLUSION: PET with 18F-fluoride demonstrates very high uptake in lytic and sclerotic breast cancer metastases.

Aged↗
PubMed1998

Interleukin-13 inhibits cell proliferation and stimulates interleukin-6 formation in isolated human osteoblasts.

Interleukin-13 (IL-13) is a recently identified cytokine that is secreted by activated T cells and regulates inflammatory responses. We have investigated the effects of IL-13 on isolated human osteoblast-like cells (hOB). IL-13 dose-dependently (1-100 pmol/L) reduced the incorporation rate of [3H]thymidine in hOB cells by more than 50%. Using a cell metabolic assay as well as direct cell counting, we found that treatment with IL-13 lead to a decrease in hOB cell number. The effect was both time and dose dependent, and after 12 days of culture, treatment with IL-13 (0.1 nmol/L) caused a 70% decrease in the number of cells. Also, IL-13 increased the levels of IL-6 messenger ribonucleic acid in hOBs, as measured by ribonuclease protection assay, and stimulated secretion of IL-6 into culture supernatants. In conclusion, IL-13 inhibits cell proliferation and increases IL-6 formation in human osteoblasts. Our findings suggest that IL-13 may cause bone loss due to impaired osteoblastic growth and IL-6-induced osteoclast recruitment.

Cell Division↗
PubMed1997

A new fluorometric assay for determination of osteoblastic proliferation: effects of glucocorticoids and insulin-like growth factor-I.

A novel fluorometric proliferation assay, AlamarBlue (AB), was used to study the proliferative capacity of isolated human osteoblasts (hOBs). AB is an oxidation-reduction indicator that yields a fluorescent signal in response to metabolic activity. The assay was performed by replacing the experiment media in a microtiter plate with a 10% AB solution and measuring fluorescence after a 3-8-hour incubation. The assay was optimized with respect to incubation time, cell density, and AB concentration. When the results of the AB assay were compared with cell counting in a Bürker chamber there were consistently good correlations (r > 0.9), regardless of the agonist with which the cells were treated. The mean intraassay coefficient of variance (CV) values were 9.9-11.8% in experiments where osteoblasts were treated for 12 days with insulin-like growth factor-I (IGF-I; 100 nM), or dexamethasone (1 micro;M). IGF-I dose dependently, at and above 1 nM, stimulated proliferation of hOBs. This effect was detectable after 3 days and reached 130-140% of untreated controls after 12 days in culture. The effects of dexamethasone (DEX) on the proliferation rate of hOBs were more complex. In short-term cultures, 3 days, DEX dose dependently stimulated proliferation. However, at and above 6 days, DEX exerted a biphasic effect, with stimulation seen at 1-10 nM and a marked inhibition of cell proliferation at and above 100 nM. dexamethasone, hydrocortisone, prednisolone, and deflazacort had almost identical biphasic effects on osteoblastic proliferation in 12 day cultures with a stimulation seen at 1-10 nM, and a marked inhibition down to 50-60% of untreated controls at and above 100 nM. When IGF-I (0. 1-100 nM; 12 day culture) was combined with different doses of DEX, IGF-I still dose dependently stimulated the proliferation rate in hOBs regardless of the amount of DEX added. The stimulatory effect of DEX (10 nM, 12 days culture) was additive to the effect of 100 nM IGF-I. We conclude that AB is an easy and reliable assay for osteoblastic cell proliferation, well suited for large scale studies of cell growth using small amounts of cells, and that IGF-I partly reverses the glucocorticoid-induced inhibition of osteoblastic proliferation.

Cell Division↗
PubMed1997

Reduced serum levels of the growth hormone-dependent insulin-like growth factor binding protein and a negative bone balance at the level of individual remodeling units in idiopathic osteoporosis in men.

Idiopathic osteoporosis in younger individuals could be related to reduced bone formation rather than increased bone resorption, and disturbances in GH or insulin-like growth factor (IGF)-I production could be involved in its pathogenesis. In the present study, men with idiopathic osteoporosis were compared with healthy men, with respect to bone histomorphometry and to serum levels of IGF-I, IGF-II, IGF binding protein (IGFBP)-2 and IGFBP-3, and 24-h urinary excretion of GH. Mean wall thickness was reduced in the patients (48.3 +/- 7.2 vs. 61.7 +/- 5.4 microns, P < 0.001). Also, resorption depth was decreased, albeit to a lesser degree (54.4 +/- 3.8 vs. 60.7 +/- 5.3 microns, P < 0.01), thus creating a pronounced negative balance (-6.04 +/- 9.8 vs. 0.96 +/- 3.2 microns, P < 0.05). In the patients, serum concentrations of IGFBP-3 were reduced, compared with controls, with a 46% lower mean value; whereas levels of IGF-I, IGF-II, IGFBP-2, and GH were similar in the two groups. Thus, there was a significant negative balance caused by a pronounced decrease in wall thickness in men with idiopathic osteoporosis. The finding of low IGFBP-3 levels in these patients is interesting, in view of previous clinical and experimental findings, but its pathophysiological significance remains to be determined.

Adult↗
PubMed1997

Regulation of interleukin-6 secretion from mononuclear blood cells by extracellular calcium.

Interleukin-6 (IL-6) is known to enhance osteoclast recruitment, and thereby bone resorption. Thus, IL-6 has been proposed to mediate hypercalcemia in multiple myeloma and the enhanced osteoclastic activity seen in postmenopausal osteoporosis. We recently reported that the calcium concentration in plasma affects IL-6 secretion from mononuclear blood cells. To investigate the underlying mechanism, we have studied the effect of calcium on IL-6 formation in mononuclear blood cells ex vivo and in vitro. Thirteen healthy volunteers were given 1 g of calcium orally after overnight fasting. Plasma levels of ionized calcium (pCa2+) and serum levels of parathyroid hormone (sPTH) were measured after 2 and 4 h, with all subjects still fasting. After 2 h, pCa2+ was increased and sPTH decreased in all 13 persons. IL-6 secretion ex vivo from mononuclear blood cells drawn 4 h after calcium intake was increased 185% as compared with IL-6 secretion from cells drawn just before calcium intake. In control experiments without calcium intake, there was no alteration in pCa2+ and no effect on IL-6 secretion from mononuclear blood cells. In vitro studies revealed that stimulation of isolated mononuclear blood cells with physiological concentrations of calcium dose-dependently increased IL-6 secretion with an estimated EC50 at 1.2 mM Ca2+. No effect on the IL-6 secretion was seen following treatment of the isolated mononuclear blood cells with PTH or calcitonin. These observations demonstrate that the plasma calcium concentration affects IL-6 secretion from mononuclear blood cells. The in vitro data indicate the involvement of a direct calcium sensing mechanism. These findings might have implications in hypercalcemia and should also be borne in mind when considering the role of cytokines in osteoporosis.

Administration, Oral↗
PubMed1997

Inflammatory cytokines regulate proliferation of cultured human osteoblasts.

We investigated the effects of various pro-inflammatory cytokines on the proliferation rate of isolated human osteoblastic cells in primary cultures. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-beta (TNF-beta) time- and dose-dependently enhanced the proliferation of human osteoblasts. Both of these cytokines also enhanced endogenous prostaglandin E2 (PGE2) formation. Exogenous PGE2 dose- and time-dependently-stimulated cell proliferation. However, the stimulatory effects of IL-1 beta and TNF-beta on osteoblast proliferation were not abolished by indomethacin, indicating a direct effect by these cytokines on the rate of proliferation. TNF-alpha stimulated proliferation at low doses, while it significantly inhibited proliferation at higher concentrations (at and above 100 pM) and with prolonged incubation times. This biphasic effect was unaffected by indomethacin. Interleukin-6, finally, did not affect the rate of proliferation. Our findings show that inflammatory cytokines may stimulate or inhibit the proliferation of isolated human osteoblasts, depending on concentration and time.

Cell Division↗
PubMed1996

Insulin-like growth factor I is a growth and survival factor in human multiple myeloma cell lines.

Human multiple myeloma (MM) represents a highly aneuploid tumor as shown by cytogenetic studies. This may partly explain the heterogeneity with regard to growth factor requirements demonstrated among MM cells. We have previously reported the expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNA in some MM cell lines. In this study we investigated the role of IGF-I as a growth and/or survival factor in three MM cell lines: LP-1, EJM, and Karpas 707. We report that all cell lines expressed IGF-I and IGF-IR mRNA and protein. LP-1 and Karpas 707, but not EJM, were stimulated to proliferation in a dose-dependent manner by exogenous IGF-I. An IGF-IR blocking antibody inhibited both the IGF-I-induced and spontaneous growth of LP-1, and Karpas 707, while the EJM cell line was unaffected by the addition of the antibody. In conclusion, our results show that IGF-I can act as a growth factor in human MM, and they suggest that an autocrine IGF-I loop may contribute to the growth and survival in some MM cell lines.

Base Sequence↗
PubMed1996

Insulin-like growth factor I does not stimulate bone resorption in cultured neonatal mouse calvarial bones.

Insulin-like growth factor I (IGF-I) has documented anabolic effects on osteoblasts, whereas its influence on osteoclasts and on bone resorption is unclear. We have investigated the effects of IGF-I on osteoclast recruitment and bone resorption in vitro. IGF-I (at and above 1 nM) stimulated the formation of multinucleated tartrate-resistant acid phosphatase positive cells in murine bone marrow cultures, incubated for 9 days. The number of multinucleated cells increased to 540 +/- 160% of control (mean +/- SEM) in cultures treated with 10 nM IGF-I. IGF-I (0.1-100 nM) had no effect by itself on 45Ca-release from prelabelled neonatal mouse calvarial bones. However, IGF-I (100 nM) had an inhibitory effect on bone resorption induced by prostaglandin E2 and 1,25(OH)2D3. These findings indicate that IGF-I enhances the formation of osteoclasts-like cells in long-term bone marrow cultures. In bone organ cultures, however, IGF-I has an inhibitory effect on stimulated bone resorption, suggesting that IGF-I inhibits existing osteoclasts and, alternatively, that IGF-I interferes with the osteoblast-derived factor(s) that stimulate existing osteoclasts.

Animals↗
PubMed1995

[Continuous remodeling of the skeleton. Growth factors and cytokines direct the activity].

Bone is living tissue perpetually undergoing metabolism in a process known as remodelling, a sequence of cellular events occurring throughout the skeleton. The process is initiated in response to bone resorption by multinucleated osteoclasts. The capacity to stimulate osteoclastic activity is a property common to a multiplicity of hormones and cytokines--e g, parathyroid hormone, vitamin D, thyroxine, interleukin-1 and tumour necrosis factor. There is also a group of growth factors and cytokines, such as interleukin-6 and interleukin-11, that serve as stimulators of osteoclastic recruitment. Following bone resorption by osteoclasts, osteoblasts are recruited to the resorption lacuna, where they secrete osteoid which is then mineralised to form mature bone. The coupling of bone resorption and formation is governed by growth factors embedded in the mineralised bone matrix and released during resorption. These include transforming growth factor beta, insulin-like growth factor. Osteoporosis is caused by imbalance between the resorption and formation phases of the remodelling cycle.

Adult↗
PubMed1995

Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption.

The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by 45Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.

Animals↗
PubMed1995

Studies on two new vitamin D analogs, EB 1089 and KH 1060: effects on bone resorption and osteoclast recruitment in vitro.

We have investigated the effects on bone resorption of two new potent antiproliferative vitamin D3 analogs, EB 1089 and KH 1060, by studying recruitment of osteoclasts in murine bone marrow cultures and 45Ca release from prelabeled neonatal mouse calvarial bones. Binding studies to vitamin D receptor protein, from human osteosarcoma MG-63 cells, demonstrated kd values of 8.5 x 10(-11) for 1 alpha,25(OH)2D3, 6.5 x 10(-11) for KH 1060, and 2.7 x 10(-10) for EB 1089. 1 alpha,25(OH)2D3 and EB 1089 were equipotent stimulators of osteoclast recruitment in murine bone marrow cultures, with EC50 at 10(-10) mol/L, whereas KH 1060 was about tenfold more potent with an EC50 at 10(-11) mol/L. In serum-free media, 1 alpha,25(OH)2D3 enhanced 45Ca release from neonatal mouse calvarial bones with EC50 at 10(-11) mol/L, but in the presence of 10% fetal calf serum (FCS) the stimulatory effect was significantly diminished, with a threshold value at 10(-10) mol/L. EB 1089 stimulated bone resorption with an estimated EC50 at 3 x 10(-11) mol/L, whereas KH 1060 was about tenfold more potent than 1 alpha,25(OH)2D3, and stimulated bone resorption with an EC50 at 10(-12) mol/L. The effects of EB 1089 and KH 1060 on 45Ca release were not significantly affected by the addition of 10% FCS. Addition of vitamin D binding protein to serum-free incubations of neonatal mouse calvarial bones, significantly inhibited the bone resorbing effect of 1 alpha,25(OH)2D3, but did not affect EB 1089 and KH 1060 induced 45Ca release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗