Sclerostin in mineralized matrices and van Buchem disease.

Sclerostin is an inhibitor of bone formation expressed by osteocytes. We hypothesized that sclerostin is expressed by cells of the same origin and also embedded within mineralized matrices. In this study, we analyzed (a) sclerostin expression using immunohistochemistry, (b) whether the genomic defect in individuals with van Buchem disease (VBD) was associated with the absence of sclerostin expression, and (c) whether this was associated with hypercementosis. Sclerostin was expressed by cementocytes in mouse and human teeth and by mineralized hypertrophic chondrocytes in the human growth plate. In individuals with VBD, sclerostin expression was absent or strongly decreased in osteocytes and cementocytes. This was associated with increased bone formation, but no overt changes in cementum thickness. In conclusion, sclerostin is expressed by all 3 terminally differentiated cell types embedded within mineralized matrices: osteocytes, cementocytes, and hypertrophic chondrocytes.

Interleukin 17 synergises with tumour necrosis factor alpha to induce cartilage destruction in vitro.

BACKGROUND: Interleukin 17 (IL17) is produced by activated T cells and has been implicated in the development of bone lesions and cartilage degradation in rheumatoid arthritis (RA). OBJECTIVE: To determine whether IL17, alone or together with tumour necrosis factor alpha (TNFalpha), induces cartilage destruction in vitro. METHODS: Fetal mouse metatarsals stripped of endogenous osteoclast precursors were used to study the effect of IL17 on cartilage degradation independently of osteoclastic resorption. Cartilage destruction was analysed histologically by Alcian blue staining. RESULTS: IL17 alone, up to 100 ng/ml, had no effect on the cartilage of fetal mouse metatarsals. IL17 (>/=0.1 ng/ml), however, induced severe cartilage degradation when given together with TNFalpha (>/=1 ng/ml). The cytokine combination decreased Alcian blue staining, a marker of proteoglycans, throughout the metatarsals and induced loss of the proliferating and early hypertrophic chondrocyte zones. TNFalpha alone also decreased Alcian blue staining, but not as dramatically as the cytokine combination. In addition, it did not induce loss of chondrocyte zones. Treatment with inhibitors of matrix metalloproteinase (MMP) activity and nitric oxide synthesis showed that MMP activity played a part in cartilage degradation, whereas nitric oxide production did not. CONCLUSIONS: IL17, together with TNFalpha, induced cartilage degradation in fetal mouse metatarsals in vitro. IL17 may, therefore, participate in the development of cartilage destruction associated with RA by enhancing the effects of TNFalpha and may provide a potential therapeutic target.

Ovariectomy and orchidectomy induce a transient increase in the osteoclastogenic potential of bone marrow cells in the mouse.

Withdrawal of gender steroids in both women and men is associated with an increase in bone turnover with bone resorption exceeding bone formation leading to bone loss. To further investigate this process, the osteoclastogenic potential of mouse bone marrow cells was assessed at different timepoints after ovariectomy (ovx) or orchidectomy (orx). Cocultures of osteoclast-free fetal mouse long bones together with bone marrow from ovariectomized or orchidectomized mice indicated that the withdrawal of gender steroids in female and male mice induces a transient increase in osteoclastogenesis. The osteoclastogenic potential of the bone marrow cells was increased 7 days after ovx or orx. However, osteoclastic resorption was not increased at 3 days after surgery and had normalized 30 days after either ovx or orx. These results suggest that the withdrawal of gender steroids induces a transient increase in osteoclastogenesis in mice of both genders, which is associated with the early phase of rapid bone loss.

Bisphosphonates inhibit the adhesion of breast cancer cells to bone matrices in vitro.

Bisphosphonates are used with increasing frequency in the management of skeletal complications in patients with breast cancer. In this paper, we have investigated whether bisphosphonates, besides their known beneficial effects on tumor-associated osteoclastic resorption, are capable of inhibiting breast cancer cell adhesion to bone matrix. For that we used two in vitro models for bone matrix (cortical bone slices and cryostat sections of trabecular bone from neonatal mouse tails). Four bone matrix-bound nitrogen-containing bisphosphonates (pamidronate, olpadronate, alendronate, and ibandronate) inhibited adhesion and spreading of breast cancer cells to bone dose-dependently, whereas etidronate and clodronate had little or no effect. Strikingly, the relative order of potency of the bisphosphonates in inhibiting the adhesion of cancer cells to cortical and trabecular bone corresponded to their relative antiresorptive potencies in vivo as well as their ranking in in vitro bone resorption assays with predictive value for their clinical efficacy. It appears that nitrogen-containing bisphosphonates alter selectively the adhesive properties of the extracellular bone matrix preventing the attachment of breast cancer cells to it. Besides the beneficial effects of bisphosphonates on tumor-induced osteoclastic resorption, the previously unrecognized effect presented in this paper makes these agents suitable for earlier pharmacologic intervention in patients with breast cancer at risk of developing bone metastases.

In vitro and ex vivo evidence that estrogens suppress increased bone resorption induced by ovariectomy or PTH stimulation through an effect on osteoclastogenesis.

The mechanism of bone loss following cessation of ovarian function is still unclear. Several studies have shown an increase in bone turnover following natural or surgical menopause which could be prevented by estrogen administration. However, a direct effect of estrogen on osteoclast-mediated bone resorption has been difficult to demonstrate in vitro. Recent evidence suggested that estrogen withdrawal stimulates the production of bone resorbing cytokines, (e.g., interleukin-6, IL-6), which regulate osteoclast formation in the bone marrow microenvironment. We studied the effects of 17beta-estradiol on osteoclastic resorption, measured as 45calcium release, in vitro using cultures of fetal mouse long bone explants in which different stages of osteoclast development and activity are represented. 17beta-estradiol (10(-12)-10(-8) M) had no effect on basal or parathyroid hormone (PTH)-stimulated resorption of bone explants in which mature osteoclasts (radii/ulnae) or osteoclast precursors/progenitors (metacarpals) are present. 17beta-estradiol, however, inhibited significantly the PTH-stimulated resorption of osteoclast-free metacarpals cultured together with mouse fetal liver as a source of early osteoclast progenitors; basal resorption was also not inhibited in this system. In ex vivo studies we further examined the effects of culturing bone marrow cells from ovariectomized (OVX) or sham-operated mice as an osteoclastic source together with osteoclast-free metacarpals on 45calcium release and bone histology. Cocultures of the bone marrow cells from OVX mice with osteoclast-free metacarpals increased significantly the osteoclast formation and subsequent osteoclastic resorption compared with control cocultures. This increase in resorption was prevented by either treatment of the OVX animals with estrogen for 1 week starting immediately after OVX or injection of the OVX animals with an IL-6 neutralizing antibody. We conclude that estrogens suppress the increased osteoclastic resorption induced by PTH or OVX through an effect on hematopoietic progenitor cells of the osteoclast lineage. Furthermore our data suggest that IL-6 is involved in the increase in osteoclastic resorption following OVX.

Effects of experimental conditions on the release of 45calcium from prelabeled fetal mouse long bones.

Embryonic/neonatal bones in culture are commonly used for the study of osteoclastic resorption in vitro. For this purpose, the release of 45calcium (45Ca) from prelabeled bones is measured as an index of resorption. We studied 45Ca release from two types of long bone explants after different preparation methods: 17-day-old fetal mouse radii/ulnae with and without cartilage ends (intact radii/ulnae and shafts, respectively), and intact 18-day old metacarpals/metatarsals. In addition, we examined the effect of different culture conditions, such as cultures performed under the surface of the medium or at the interphase of medium and air, on 45Ca release and histology. When intact radii/ulnae were cultured under the surface of the medium, there was always a significant amount (10%) of net basal 45Ca release (corrected for physicochemical exchange) that was not due to osteoclastic resorption, as it could not be suppressed by inhibitors of resorption even at high concentrations. Moreover, histologically TRAcP-positive cells were almost absent after culture and the bone marrow/stromal cells in the center of the bone appeared necrotic, possibly due to a lack of oxygen. Under these culture conditions, osteoclasts could survive in shafts as well as in PTH-stimulated intact radii/ulnae, but a constant amount of 10% 45Ca, not due to resorption, was still released in the medium. When these explants were cultured at the interphase of medium and air, basal and stimulated 45Ca release originated from osteoclastic resorption. In contrast, in 18-day-old fetal mouse metacarpals/metatarsals, the experimental conditions applied did not affect 45Ca release, which was always due to resorption of the explants by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of a rat model for the simultaneous assessment of pharmacokinetic and pharmacodynamic aspects of bisphosphonate treatment: application to the study of intravenous 14C-labeled 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonate.

Bisphosphonates are drugs that suppress osteoclast-mediated bone resorption and are used with increasing frequency in the treatment of skeletal disorders. Therapeutic regimens are largely based on pharmacodynamic information because of difficulties in obtaining and interpreting pharmacokinetic data. We describe here the application of a permanently cannulated rat model, previously used in other areas of endocrine research, to the simultaneous study of pharmacokinetic and pharmacodynamic properties of the newly developed bisphosphonate EB-1053 [1-hydroxy-3-(1-pyrrolidinyl)propylidene-1,1-bisphosphonate]. Two groups of five rats each received daily intravenous injections of [14C]EB-1053 (0.025 and 0.1 mg/day, respectively); a third group (n = 7) received only normal saline injections and served as control. Treatment was given for at least 20 days. A fourth group (n = 3) received IV injections of the bisphosphonate on three separate occasions. Following IV administration, EB-1053 was rapidly cleared from the circulation. Urinary excretion of radioactivity reached about 55% of the daily administered dose within 48 h and remained at this level during the whole treatment period, indicating continuing retention of the bisphosphonate. Bone resorption, assessed biochemically as the hydroxyproline to creatinine ratio in urine, was suppressed effectively with both doses used. Suppression reached a maximum around day 4 and remained at the same level until the end of treatment. Accumulation of the bisphosphonate in the skeleton was therefore not associated with a cumulative effect on bone resorption. This strongly suggests that in treatment planning a distinction should be made between surface-bound and hence biologically active bisphosphonate from the drug which is incorporated in bone during bone turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukemia inhibitory factor inhibits osteoclastic resorption, growth, mineralization, and alkaline phosphatase activity in fetal mouse metacarpal bones in culture.

Leukemia inhibitory factor (LIF) has been reported to affect bone metabolism, but results are variable. We examined the effect of mouse recombinant LIF on osteoclastic resorption in fetal bone explants representing different stages of osteoclast development. In cultures of 17-day-old fetal mouse metacarpals in which only osteoclast progenitors and precursors are present, resorption (measured as 45Ca release) was significantly inhibited to 29.2% and to 96.6% in the presence of LIF 100 and 1000 U/ml, respectively. Histologic examination of the explants treated with 1000 U/ml of LIF confirmed the biochemical findings and showed that osteoclast progenitors and precursors remained in the periosteum and did not invade the mineralized matrix. In metacarpals of older fetuses (18- and 19-day-old) in which the mineralized cartilage has been invaded by mature osteoclasts, the inhibition of resorption by LIF (1000 U/ml) was 87.9 and 74.7%, respectively, the latter being significantly less than the inhibition observed in 17-day-old metacarpal cultures. The inhibitory effect of LIF was absent during concurrent administration of PTH or 1,25-(OH)2D3 and could be reversed by PTH. In addition, LIF was found to inhibit growth, mineralization, and alkaline phosphatase activity in metacarpals independently of osteoclastic resorption. These results suggest that LIF affects the development rather than the activity of osteoclasts, probably through an effect on the osteogenic cells. LIF may be an important endogenous regulator of bone metabolism.

Disodium 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonate (EB-1053) is a potent inhibitor of bone resorption in vitro and in vivo.

The ability of the new nitrogen-containing bisphosphonate disodium-1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphona te (EB-1053) to inhibit osteoclastic resorption was examined in vitro and in vivo. Results were compared to those obtained with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (pamidronate or APD). In vitro, when tested in osteoclast precursor-dependent systems (fetal mouse metacarpals and a coculture system), EB-1053 suppressed 45Ca release effectively and was found to be about 10 times more potent than pamidronate (ED50 = 2.5 x 10(-7) versus 2.5 x 10(-6) M, respectively). The EB-1053-inhibited osteoclastic resorption could be reversed by treatment with parathyroid hormone (PTH). In vivo, daily subcutaneous injections of EB-1053 to young growing rats for 7 days increased metaphyseal bone mass in tibiae dose dependently. In these experiments EB-1053 was about 50 times more potent than pamidronate. These studies show that EB-1053 is a very potent bisphosphonate that has potential use in the treatment of skeletal disorders.

Modulation of PTH-stimulated osteoclastic resorption by bisphosphonates in fetal mouse bone explants.

We examined the effects of the bisphosphonates Cl2MDP, APD, and Me2APD on osteoclastic resorption in the absence and presence of PTH using fetal mouse osteoclast-free bone explants cocultured with fetal liver as a source of osteoclast precursors. Results revealed qualitative and quantitative differences among the bisphosphonates tested. With Cl2MDP and APD fractional inhibition of resorption (measured as 45Ca release) in the presence of PTH was proportional to that obtained in its absence. In contrast, Me2APD, which is the most potent inhibitor of the three, was found at low concentrations (less than or equal to 5 x 10(-7) M) to enhance the PTH-stimulated osteoclastic resorption. APD as well, at concentrations that could not inhibit resorption, had a similar effect, but Cl2MDP did not. These studies describe a new phenomenon, that low doses of nitrogen-containing bisphosphonates can act synergistically with PTH and enhance osteoclastic resorption. These findings may have clinical implications in the management of patients with increased osteoclastic resorption due to parathyroid overactivity.

Two distinct effects of recombinant human tumor necrosis factor-alpha on osteoclast development and subsequent resorption of mineralized matrix.

The multifunctional cytokine tumor necrosis factor-alpha (TNF alpha) stimulates osteoclastic resorption. It is not known which steps in osteoclast formation are affected by TNF alpha. We have investigated the effects of recombinant human TNF alpha (rhTNF alpha) on osteoclast development and osteoclastic resorption in two different in vitro resorption systems which are each characterized by a different stage of development of the osteoclast. The effects were further compared to those of bovine PTH-(1-84). rhTNF alpha at concentrations between 0.01-50 ng/ml (3 x 10(-13) to 1.5 x 10(-9) M) did not alter the activity of mature osteoclasts, measured as 45Ca release in fetal mouse radii. In the osteoclast precursor-dependent system (fetal mouse metacarpals) rhTNF alpha had a biphasic effect. It stimulated resorption dose-dependently from 0.01 ng/ml onward, with a maximal response at 0.5 ng/ml. At concentrations above 10 ng/ml rhTNF alpha, resorption was inhibited. In experiments in which irradiation was used to block replication, it was found that TNF alpha stimulates the proliferation of osteoclast progenitors at both low and high concentrations. As a result, at relatively low concentrations, more osteoclasts were formed in the calcified matrix, coinciding with an increased release of 45Ca. However, at relatively high concentrations, the increase in osteoclast progenitors did not lead to increased resorption, since the putative osteoclast progenitors were arrested in the periosteum. In comparison, bovine PTH-(1-84) stimulated resorption independent of proliferation by enhancing the differentiation of postmitotic osteoclast precursors and activating mature osteoclasts. In conclusion, the effects of TNF alpha on osteoclastic resorption are dependent on the stage of osteoclast development and the concentrations applied.

Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate.

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.

Enhancement of the inhibitory action of APD on the transformation of osteoclast precursors into resorbing cells after dimethylation of the amino group.

The amino-bisphosphonate APD is distinct from the bisphosphonates EHDP and Cl2MDP by a greater molar potency in vivo as inhibitor of osteoclastic bone resorption and in vitro by a pronounced inhibitory effect on the accession of osteoclast precursors to mineralized matrix. Dimethylation of the aminogroup, which increases the basic properties of this residue but precludes others, like the liability to glucuronidation or acetylation, increased the in vivo potency of this amino bisphosphonate, as well as its in vitro specificity for osteoclast-precursor accession, but decreased its cellular toxicity. The in vitro actions of dimethyl-APD were reversible with administration of PTH. It is concluded that the introduction of a basic residue in bisphosphonates may increase affinity for the specific sites on the mineralized matrix that are involved in directing the accession of precursors and their transformation into actively resorbing osteoclasts.

Two modes of action of bisphosphonates on osteoclastic resorption of mineralized matrix.

Pretreatment of a long bone explant with P-C-P can prevent the osteoclastic resorption of its mineralized matrix, when it is entirely dependent upon activation and accession of extra-osseous osteoclast precursors. When treatment of the explant is postponed until after the development of mature osteoclasts, the P-C-P dose required for an inhibitory effect is increased 100-fold for the amino bisphosphonate APD, but not for EHDP and Cl2MDP. It is concluded that high doses of all P-C-Ps inhibit the resorbing osteoclast, but that low dose of the amino P-C-P can specifically inhibit the accession of osteoclast precursors to mineralized matrix. Both actions require P-C-P binding to the mineral. The relative potencies of the P-C-Ps in the precursor-dependent system correspond to their relative potencies in vivo. This suggests that inhibition of accession underlies the high potency which the aminobisphosphonate has in vivo.

Apposition and resorption of bone during oral treatment with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD).

The effects of 1.5-2 years oral administration of disodium (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) on bone metabolism were studied in male and female rats. APD was mixed in the food at levels of 500, 2,000 and 10,000 ppm. A dose-dependent increase in metaphyseal bone was found, indicative of continued inhibition of bone and cartilage resorption. APD did not affect mineralization of bone and cartilage, primary bone formation, or periosteal apposition. A short-term metabolic balance study was performed to compare the effects of oral with subcutaneous APD. Absorption of APD was in the order of 0.2%. Oral APD increased absorption of phosphate, probably by complexation of calcium with APD. The excess absorbed phosphate increased phosphaturia and decreased urinary calcium.

Kinetic studies of bone and mineral metabolism during treatment with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) in rats.

Dose-related effects of APD on bone metabolism and Ca homeostasis were studied in rats. The experimental approach consisted of longitudinal and cross-sectional observations, aiming at a kinetic interpretation. Bone and cartilage resorption was inhibited with 2--8 days at doses between 0.16 and 16 mumol/kg body weight/day. This was followed by changes in bone apposition that needed at least 23 days for a maximal effect. The time lag created a transient dissociation between resorption and apposition resulting in excess Ca and P retention, adding to increased metaphyseal bone mass. At high doses of APD (greater than or equal to 40 mumol/kg/day)the mineral content of new matrix decreased, associated with impairment of longitudinal growth of long bones. It is concluded that the lower doses of APD inhibited resorption of bone and cartilage, possibly by physicochemical stabilization of bone mineral, whereas the effect on bone apposition was due to a cellular homeostatic mechanism. Inhibition of growth and of matrix calcification, requiring much higher doses, may be due to a direct, toxic effect on bone cells. The modes of action of APD are discussed in relation to EHDP and Cl2MDP.