Controlled release of bisphosphonate from a calcium phosphate biomaterial inhibits osteoclastic resorption in vitro
Corinne FaucheuxÉlise VerronAssem SoueidanSolen JosseM. ArshadPascal JanvierPaul PiletJean‐Michel BoulerBruno BujoliJérôme Guicheux
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Abstract Calcium phosphate biomaterials such as calcium deficient apatite (CDA) have been contemplated as carrier for delivery of bisphosphonate in bone tissues. In the present work, we have investigated the in vitro biological properties of Zoledronate‐loaded CDA. CDA was loaded with zoledronate according to a previously described coating process. 31 P MAS NMR spectra demonstrated the effective loading of zoledronate onto CDA. Using 14 C labeled zoledronate, we then demonstrated the in vitro release of zoledronate from CDA. In a first set of experiments, we confirmed that Zoledronate reduced the number of TRAP‐, vitronectin receptor‐, and F‐actin ring‐positive cells as well as the resorption activity of osteoclasts obtained from a total rabbit bone cell culture. Interestingly, Zoledronate‐loaded CDA and its extractive solutions decreased the osteoclastic resorption. Finally, zoledronate‐loaded CDA did not affect the viability and alkaline phosphatase activity of primary osteoblastic cells. These data demonstrate that CDA is effective for loading and release of zoledronate. The released zoledronate inhibited osteoclastic resorption without affecting osteoblasts. Our findings therefore suggest that such a drug delivery system would allow an increase in the efficiency of bisphosphonates by being locally available. Further experiments are now required to evaluate the in vivo antiresorptive activity of this concept. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009Abstract It has previously been reported that low extracellular pH stimulates the excavation of resorption lacunae by rodent osteoclasts in vitro. Using avian bone cells in a similar in vitro assay we have demonstrated that osteoclast activity is optimal at pH 7.20–7.40 and is inhibited at extremes of pH (<7.10 and >7.60). Over the first 24 h of incubation at low pH there may be an increase in osteoclastic resorption but to a lesser extent than that reported for rodent cells. However, after 24–30 h in culture there is little or no further increase in bone resorption, presumably due to a cytotoxic effect of low pH acting either on the osteoclast directly or via nonosteoclastic bone cells. In contrast to a previous report, in which preincubation of wafers for 24 h had no effect on bone resorption, we found that preincubation of bone substrates at pH 6.50 for longer periods enhances subsequent resorption at pH 7.20.
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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.
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A new bone resorption model was developed by using living bone substrates and devitalized bones for isolated osteoclasts to act on. The extent of bone resorption was assessed by measuring the area and depth of resorption pits. The area and depth of pits made on living bones were greater than those of pits made on devitalized bone substrates. TIMP (100 micrograms/ml) reduced resorption on living bone in area and depth to the same amount of resorption on devitalized bone. E-64 (60 microM) significantly inhibited the resorption of devitalized bones. TGF-alpha (100 ng/ml) did not have significant effect on the resorption of any substrate. Indomethacin (100 ng/ml) reduced resorption on living bone to the same level of that on devitalized bone. These results suggest that resorption on living bone is aided by osteocyte-synthesis of metalloproteinases, among them collagenase, to degrade bone collagen through prostaglandin synthesis by viable cells in the substrates. The stimulation of bone resorption by TGF-alpha observed in organ culture appears not to be mediated by direct stimulation of osteoclast activity.
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Palytoxin, a nonphorbol ester-type tumor promoter, stimulated the production of prostaglandin E2 (PGE2) and bone resorption in neonatal mouse calvariae in organ culture. The action of palytoxin on bone resorption occurred at extraordinarily low concentrations; enhanced resorption was regularly observed at 0.5 pg/ml, and the ED50 was 1-2 pg/ml (-3 × 10-13 M). Palytoxin-induced formation of PGE2 and bone resorption were inhibited completely by indomethacin (200 ng/ ml). Concentrations of palytoxin above 10 pg/ml led to progressively decreasing enhancement of bone resorption; by 100–250 pg/ml no stimulation of resorption was observed despite continued high production of PGE2. Treatment with high concentrations of palytoxin (100 or 250 pg/ml) for 24–72 h inhibited cAMP accumulation stimulated by exogenous PGE2 or PTH and inhibited bone resorption induced by PGE2, PTH, or an analog of cAMP. Thus, palytoxin exhibited a biphasic doseresponse curve for enhanced bone resorption, with stimulation at low concentrations (0.5–10 pg/ml) and toxic inhibition at high concentrations (>50 pg/ml). Palytoxin is one of the most potent stimulators of bone resorption yet identified. (Endocrinology120: 1338–1345,1987)
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In this study, a new bone resorption model was developed by using living bone substrates prepared from mice calvaria. Devitalized bones made by freezing and thawing of living bones as well as dead cortical bone slices of monkey femur were also used as substrates for isolated osteoclasts to act upon. The extent of bone resorption was assessed by measuring both the area and the depth of resorption pits. Recombinant Human Tissue Inhibitor of Metalloproteinases (TIMP) and Cysteine-Proteinase Inhibitor (E-64) were used to find out the different mode and extent of resorption by isolated osteoclasts between living and devitalized bones. Transforming Growth Factor-α(TGF-α) was applied to this model to investigate the possible mechanisms involved in bone resorption induced by tumors.Both the area and the depth of pits made on living bones were more extended compared to those of pits made on devitalized bone substrates. TIMP (100μ/ml) reduced the resorption of living bone both in the area and in the depth to the same amount of resorption made on devitalized bone. But, TIMP did not inhibit the resorption of devitalized bone. E-64 (60μM) significantly inhibited the resorption of devitalized bones by 66%, while it had no effect on the resorption of living bone. TGF-α(100 ng/ml) did not show significant effect on the resorption of any substrate, but appeared to inhibit the osteoclast activity slightly (p<0.1). Indomethacin (100 ng/ml) reduced the resorption of living bone to the same level of that of devitalized bone, but did not, however, inhibit the resorption of devitalized bone. These results suggest that:1 The resorption of living bone appeared to be aided by osteocyte-synthesis of metalloproteinases, among them collagenase, to degrade bone collagen.2. The excess of resorption of living bone compared to that of devitalized bone also depended on prostaglandin synthesis by living cells, interior to the substrate.3. Thus, the mechanisms of resorption underlying living bone and devitalized bone are different.4 TGF-α(100 ng/ml) appeared to inhibit the activity of isolated osteoclast, but not significant (13, 1, 0.1). So, the stimulation of bone resorption by this factor observed in vivo may be caused first by living cells other than osteoclast.5. This new culture system of isolated osteoclasts using living bone substrate presents a useful model for the study of bone resorption.
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Objective: To compare the function of bone resorption by the multinucleated giant cell (MGC) separated from giant cell tumour(GCT) with the osteoclast. Methods: The multinucleated giant cells (MGC) were separated from giant cell tumour and cultured with bone slice. The effect of IL-1α(50 μg/L, 100 μg/L)on bone resorption by MGC was observed. Results: IL-1α enchanced the numbers and the area of bone resorption lacunae on bone slices and it was dosedepended. MGC could maintain the ability of bone resorption over a long period(1 month). Conclusion: IL-1α promotes bone resorption induced by MGC. The MGC does have the same function of bone resorption as the osteoclast.
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The bisphosphonates (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) and disodium dichloromethylidene bisphosphonate (Cl2MDP) effectively inhibit the accelerated bone resorption associated with some skeletal disorders, e.g., Paget's disease. However, it has not been established whether these compounds exert their inhibitory effect by rendering the bone mineral more resistant to degradation, by diminishing the activity of resorbing cells, or through some combination of both activities. In this study, we have tested these possibilities using an in vitro resorption assay system consisting of elicited rat peritoneal macrophages co-cultured with particles of 45Ca-labeled, devitalized rat bone. This assay system permits the quantitative assessment of the action of APD and Cl2MDP on the two major phases of bone resorption (cell-substrate attachment and osteolysis) under circumstances where the drugs are present continuously or, most importantly for the issues in question, after the separate pretreatment of the particles or the resorbing cells.
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The incubation of isolated osteoclasts with devitalized bone has become a widely used method for the recent study of bone resorption. Although the studies employing this method have definitively demonstrated that isolated osteoclasts have an avid capacity to resorb devitalized bone, the resorption in this model appears to be different from that of living bone as observed in vivo and in organ culture studies. To evaluate how the resorption of living bone is different from that of devitalized bone, we have extended this bone resorption model using isolated osteoclasts by including both devitalized and living bone substrates. Living bone substrates were freshly prepared from calvaria of 8- to 12-month-old mice. Periosteum, cellular components, and osteoid were completely scraped off to leave a rigid, smooth, mineral-exposed surface for the isolated osteoclasts to act upon. Some of the bone pieces were devitalized by repeated freezing and thawing. Living and devitalized bones were cultured with isolated rabbit osteoclasts for 60 h with or without recombinant human tissue inhibitor of metalloproteinases (100 micrograms/ml). The extent of bone resorption was assessed by measuring both the area and the depth of resorption pits. Comparing the areas of the resorption pits showed significantly more resorption in living bone than in devitalized bone (27% of that of living bone). Recombinant human TIMP reduced the resorption of living bone by 73% but did not, however, inhibit the resorption of devitalized bone. Similarly, resorption pits formed on the living bones were significantly deeper (on the average, 12.4 microns) than those formed on the devitalized bones (on the average, 4.3 microns). The average depth of the resorption pits on living bone was significantly reduced by the presence of the inhibitor, whereas there was no difference between the control and inhibitor-treated devitalized bones. These results suggest that the mechanisms underlying the resorption of living bone and that of devitalized bone are not the same and that the resorption of living bone is aided by osteocytes.
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