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Effects of sodium acetate on rat bone-nodule formation and mineralization in vitro
ARCHIVES OF
PERGAMON
Archives of Oral Biology 43 (1998) 729±733
ORAL BIOLOGY
Eects of sodium acetate on rat bone-nodule formation and mineralization in vitro L.A. Visconti a, E.H.K. Yen a, b, R.B. Johnson c, * a
Department of Preventive Dental Science, University of Manitoba, Winnipeg, Manitoba, Canada Dean, University of British Columbia Faculty of Dentistry, Vancouver, British Columbia, Canada c Department of Diagnostic Sciences, University of Mississippi Medical Center, Jackson, MS 39216-4505, USA b
Accepted 20 February 1998
Abstract Sodium acetate reportedly promotes bone atrophy by inducing resorption and inhibiting osteoprogenitor-cell proliferation, but little is known about its eects on bone-matrix deposition and mineralization by a population containing osteoprogenitor cells. The objective here was to assess the eects of 1±20 mM sodium acetate on the proliferation and dierentiation of these cells and their resultant bone-nodule formation and mineralization in an in vitro assay. Exposure to 10 mM sodium acetate had no eect on cellular proliferation but signi®cantly increased the production and mineralization of bone nodules ( p < 0.01), suggesting that it aected osteoprogenitor dierentiation and subsequent metabolism. However, 10 mM acetate did not increase net bone mass. Dilutions of 1±5 and 20 mM inhibited cellular proliferation and resultant bone-nodule formation and mineralization, signi®cantly reducing the percentage bone area as compared to controls ( p < 0.001). These data suggest that 1±5 and 20 mM sodium acetate signi®cantly inhibit bone deposition, whereas 10 mM has no eects, which could contribute to iatrogenic metabolic bone disease in patients receiving either renal dialysis or total parenteral nutrition. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Sodium acetate; Bone matrix; Bone mineralization; Osteoblast; Osteoporosis
1. Introduction Patients on renal dialysis and those receiving total parenteral nutrition have a high probability of concurrently developing metabolic bone disease. Substitution of acetate for bicarbonate in haemodialysis ¯uids was proposed to simplify the procedure by eliminating the need for a separate pump for bicarbonate concentrate (Mion et al., 1964). Acetate (35 mM) is included in more than 90% of haemodialysis in the United States and United Kingdom (Veech, 1986). Toxic eects of acetate occur when it is converted to bicarbonate, which promotes the intracellular accumulation of cal-
Abbreviations: IGF-1, insulin-like growth factor-1, PBS, phosphate-buered saline, TCA, trichloroacetic acid. * Corresponding author.
cium, phosphate and pyrophosphate resulting in secondary hyperparathyroidism and eventual osteoporosis (Veech, 1986). In addition, the high concentrations of intracellular inorganic phosphate will chelate metals (such as aluminium) and exacerbate their cytotoxicity on brain and bone cells (Alrey et al., 1980; Prior et al., 1982; Sherrard et al., 1985). There has been little support for the idea of removing sodium acetate from these solutions, as it is a relatively inexpensive and eective buer and complications from its use are not considered signi®cant. Despite controversy over the relation between the serum acetate and metabolic bone disease, there are few studies on its eect on either the life-cycle or the metabolism of the osteoblast. In an in vitro study of chick osteoprogenitor cells, Saitta et al. (1989) reported that 5±20 mM sodium acetate decreased their rate of proliferation while increasing their alkaline phospha-
0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 8 ) 0 0 0 2 7 - 2
730
L.A. Visconti et al. / Archives of Oral Biology 43 (1998) 729±733
tase activity. However, that study was short and could not predict whether the resultant bone deposition or mineralization would be altered. There is no published information on the direct eects of sodium acetate on bone-matrix deposition and mineralization, although one in vitro study of fetal femurs showed that acetate decreased the dry weight of the bones, suggesting its use as a buer in solutions for total parenteral nutrition could promote bone atrophy (Saitta et al., 1989). Thus, it is worthwhile to investigate further the eects of acetate on the proliferation and metabolism of osteoprogenitor cells with the aim of acquiring additional information for designing preventive methods to minimize iatrogenic bone loss during renal dialysis or total parenteral nutrition. 2. Materials and methods 2.1. Explant procedure The procedure for the isolation of osteogenic cells was a modi®cation of two previously described techniques (Ecarot-Charrier et al., 1983; Beresford et al., 1984). Parietal bones were removed aseptically from 21-day-old embryos of female Sprague±Dawley rats. The periosteum was removed and the bones washed three times with PBS containing 1% antibiotics. Bones were then cut into fragments of 3±5 mm dia. and incubated in a collagenase solution [3 mg/ml type IA bacterial collagenase (from Clostridium histolyticum, collagen digestion activity = 550 units/mg; Sigma)] for 10 min at 378C to remove the ®broblast population. They were then washed with fetal bovine serum to inhibit enzymatic activity. Bone pieces (0.2±0.6 g) were placed in 60-mm tissue-culture dishes and incubated for 1 h at 378C to promote their adherence to the bottom of the dish. Growth medium (2 ml of a-minimal essential medium, pH 7.2, containing 2.2 g/l sodium bicarbonate + 15% fetal calf serum + 10% antibiotics; Sigma) was added to each dish and a further 2 ml of that medium added after 24 h. Growth medium was changed every second day thereafter. The cells reached con¯uence after 7 days. They were then released with trypsin±EDTA (1 ; Sigma), and plated in 35-mm tissue-culture dishes at a density of 2.5 104 cells/dish. 2.2. Cell isolation and growth Osteogenic cells were grown in vitro essentially by the techniques of Bellows et al. (1986): 3 104 cells were plated in 35-mm dishes and maintained in either standard medium [a-minimal essential medium supplemented with 1% antibiotics, 15% heat-inactivated fetal bovine serum (Sigma), 50 mg/ml ascorbic acid (Spindler et al., 1989), 10 mM sodium b-glyceropho-
sphate (Gronowicz et al., 1989) and 1 10ÿ7 M dexamethasone (Sigma) (Bellows et al., 1987)] or test medium, which was a modi®ed standard medium with 1, 3, 5, 10 or 20 mM sodium acetate substituted for equimolar amounts of sodium bicarbonate; this substitution does not diminish the buering capacity of the medium (Saitta et al., 1989). Some cultures were exposed to 10ÿ7 M human recombinant IGF-1 (Sigma) (Hock et al., 1988). The pH of the medium was adjusted to 7.4 before contact with the cells. Dishes were maintained at 378C in a humidi®ed atmosphere consisting of 95% air and 5% CO2. Medium was changed every 3 days. 2.3. Determination of cell proliferation The eect of sodium acetate on the proliferation of cells was determined by whole-cell counts and by evaluation of the incorporation of [3H] thymidine into DNA. During the ®nal 24 h of incubation, growth medium was replaced with serum-free medium. During the ®nal 2 h cells were exposed to [6ÿ3H] thymidine (1 mCi/ml spec. act., 29 Ci/mmol; Amersham Life Science). Medium was removed and cells were exposed to trypsin±EDTA (1 ; Sigma) for 5 min, the remaining adherent cells removed by scraping, and a 0.5-ml portion counted in a Coulter counter. A volume (1 ml) of the remaining cell suspension was centrifuged, the supernatant decanted, and the cells resuspended in 10% TCA for 20 min and centrifuged. This procedure was repeated. The TCA was decanted and the cells were solubilized in 0.5% sodium hydroxide for 60 min at 708C. The radioactivity was determined from a 100ml portion of the cell extract by liquid-scintillation analysis (Farley et al., 1988). Data were expressed as dis/min per 105 cells. 2.4. Bone-nodule formation The eects of sodium acetate on cell proliferation and resultant bone-matrix formation were determined in the nodule assay described by Bellows et al. (1986). Osteogenic cells were maintained for 21 days in medium supplemented with sodium acetate or 10ÿ7M IGF1, as previously described. At the end of the culture period, cells were ®xed with 10% neutral-buered formalin and bone nodules stained by the von Kossa method for calcium (Luna, 1968). Images of each culture were captured on computer disk and the nodules were counted and the percentage bone area calculated by densitometry using an image-analysis program [Microcomputer Imaging Device (MCID), Imaging Research; St. Catharines, Ontario]. Data were expressed as the number of bone nodules per culture and the percentage of the dish occupied by bone (percentage bone area).
L.A. Visconti et al. / Archives of Oral Biology 43 (1998) 729±733
2.5. Determination of bone-nodule mineralization The eects of sodium acetate on bone-nodule mineralization were determined by measurement of 45Ca uptake into bone nodules, as described by Bellows et al. (1991) and Ellies et al. (1993). Cells were plated as previously described and were maintained for 18 days in standard medium (containing 0, 5, 10, 20 mM sodium acetate or 10ÿ7M IGF-1) without b-glycerophosphate, followed by a 3-day incubation in pulsechase medium [standard medium supplemented with 10 mM sodium b-glycerophosphate and 0.1 mCi 45Ca/ ml (spec. act. 50 mCi/mg Ca; Amersham)]. Phase-contrast photomicrographs were taken of each culture, images captured on a computer disk, and the percentage bone area measured by densitometry using the MCID image-analysis program. Following this incubation, cell layers were washed twice with PBS and exposed to 10% formic acid for 24 h. 45Ca radioactivity was assessed by liquid-scintillation analysis. Background counts were made from cultures that had been exposed to 45Ca, but not b-glycerophosphate, and were subtracted from counts in treatment groups in all experiments. Data were expressed as dis/min per mm2 to normalize 45Ca incorporation to nodule mass. 2.6. Statistical analysis Means were calculated for each group and number of nodules and dis/min compared by factorial analysis of variance and Shee comparisons. Percentages were compared by the Kruskall±Wallis non-parametric
731
ANOVA and Dunn's multiple-comparison test. Dierences were considered signi®cant when p < 0.05.
3. Results All concentrations of sodium acetate promoted cell growth and mean cell numbers were higher in most cultures exposed to sodium acetate after day 1. At day 3 the incorporation of [3H] thymidine into DNA peaked in all groups except those with 5 and 10 mM acetate, which peaked after 5 days of incubation. Most concentrations of sodium acetate inhibited [3H] thymidine incorporation into DNA as compared to control or IGF-1 during days 3±7 of culture (Table 1). Wholecell counts at day 7 were signi®cantly lower in cultures exposed to 1±5 and 20 mM concentrations of sodium acetate; exposure to 10 mM produced no changes in cell numbers (Table 1). Sodium acetate at concentrations of 1±5 mM and 20 mM signi®cantly inhibited the percentage bone area, while 10 mM concentrations increased that area as compared to control or IGF-1 ( p < 0.001) (Table 2). However, no concentration of sodium acetate increased the percentage bone area compared to control values (Table 2). Sodium acetate at concentrations of 1±5 and 20 mM inhibited 45Ca incorporation into bone nodules ( p < 0.05); 10 mM sodium acetate enhanced radioisotope incorporation but was not signi®cantly more eective than IGF-1 (Table 3).
Table 1 Eects of various dilutions of sodium acetate (0±20 mM) and 10ÿ7M IGF-1 on [3H] thymidine incorporation (dis/min per 105 cells) into osteogenic cells 1±7 days after plating mM 0 1 3 5 10 20 IGF-1
Days 1
3 d
10.1620.81 (0.5120.08) 10.1420.87d (0.9320.13b,f) 9.442 0.32d (0.8320.09b) 4.922 0.84b,d (1.0620.14b,f) 3.582 0.29a,d (0.8620.12c) 0.352 0.03a,d (0.9220.12b,f) 26.4222.75a (0.6820.04)
5 d
53.672 3.34 (6.3720.83) 39.082 5.10c,d (6.0220.78) 28.422 13.35a,d (3.9920.56) 17.292 0.72a,d (2.7120.35b,e) 15.572 0.36a,d (6.8120.89) 0.8620.11a,d (0.9920.15a,d) 139.502 15.71a (4.6820.34)
Numbers in parentheses indicate cells 10ÿ5; n = 12 for each group. Signi®cantly dierent from O (control): ap < 0.001, bp < 0.01, cp < 0.05. Signi®cantly dierent from IGF-1: dp < 0.001, ep < 0.01, fp < 0.05.
7 d
16.1322.19 (10.3621.45) 1.9120.08a,d (7.4120.89) 1.5220.06a,d (4.5420.68b,e) 27.3620.30a,e (4.0120.61b,e) 45.9120.57 (8.9921.25) 0.3620.03a,d (1.3520.16a,d) 43.8425.61a (9.7820.93)
0.99 20.02 (12.822 2.27) 0.36 20.02a,f (7.302 0.87b,d) 0.29 20.05a,f (5.132 0.77b,d) 1.16 20.20 (8.072 1.29c,e) 0.56 20.11c (11.032 1.76) 0.2 20.03a,f (2.562 0.33a,d) 1.18 20.31 (15.492 2.01)
732
L.A. Visconti et al. / Archives of Oral Biology 43 (1998) 729±733
Table 2 Mean number of bone nodules and percentage bone area (2SEM) produced by cells exposed to various dilutions of sodium acetate or 10ÿ7M IGF-1 Bone Concentration (mM) nodules (No.) 0 (Control) 1 3 5 10 20 IGF-1
% area
e
32.2122.96f 14.1122.48a,d 12.6821.88a,d 15.6122.56b,d 21.0022.92c,e 4.1520.53a,d 48.8424.93a
31.4021.97 15.0021.54a,d 20.3021.58a,d 18.3621.14a,d 49.9223.42a,d 4.6421.28a,d 47.1025.21a
n = 12 for each group. Signi®cantly dierent from control: c p < 0.05. Signi®cantly dierent from IGF-1: f p < 0.05.
a
p < 0.001,
d
p < 0.001,
b
p < 0.01,
e
p < 0.01,
4. Discussion Sodium acetate is a compound found in many solutions for total parenteral nutrition and in some renaldialysis buers. It disrupts normal bone turnover by either producing secondary hyperparathyroidism (Veech, 1986) or inhibiting osteoprogenitor-cell proliferation (Saitta et al., 1989). To date, we believe only one study of the eects of sodium acetate on osteoprogenitor cells has been published (Saitta et al., 1989). That study was short (48 h) and reported the eects of the agent on the proliferation and not the progression phase of the life-cycle of the osteoblast. Long-term culture (15±21 days) of rat calvarial cells in the presence of ascorbic acid and sodium-b-glycerophosphate results in the formation of discrete, three-dimensional bone nodules. The number and size of these nodules can be quanti®ed and are subject to control by various agents (Henderson and Johnson, 1995; Johnson and Henderson, 1997). A nodule results from the proliferTable 3 45 Ca incorporation into bone nodules by cells exposed to various dilutions of sodium acetate or 10ÿ7M IGF-1 Concentration
Dis/min per mm2
0 1 3 5 10 20 IGF-1
3648.212412.42 1161.672127.78a,d 984.382147.75a,d 1143.982274.56a,d 6815.0721090.42b 336.43226.91a,d 4786.322622.18
n = 12 for each group. Signi®cantly dierent from control: ap < 0.001, bp < 0.01. Signi®cantly dierent from IGF-1: dp < 0.001.
ation and dierentiation of a discrete class of osteoprogenitor cells present at a low frequency in the population (Bellows and Aubin, 1989). Our study suggests that sodium acetate has adverse eects on the proliferation of that osteoprogenitor population and the deposition and mineralization of its matrix. Thus, it could potentially have adverse eects on both the deposition and resorption of bone in vivo. Renal-dialysis ¯uids contain 35±40 mM acetate (Saitta et al., 1989); ¯uids for total parenteral nutrition contain 48±146 mM acetate (Veech, 1986). Normal serum acetate is 0.01±0.4 mM (Skutches et al., 1979; Mansell and Wing, 1983) and many become routinely elevated to 3±5 mM after dialysis (Gonzalez et al., 1974; Novello et al., 1976; Mansell and Wing, 1983); concentrations exceeding 15 mM have been reported in patients on haemodialysis. Patients receiving total parenteral nutrition probably have a higher serum acetate because the solutions contain more acetate than in renal-dialysis ¯uids. Thus, the concentrations of acetate that we chose to test in vitro are similar to those in a medically compromised population. We found that 10 mM concentrations of sodium acetate increased bone-nodule formation and mineralization, whereas all other concentrations tested inhibited these functions. Agents such as sodium ¯uoride produce their maximal biological eects on osteogenic populations at concentrations close to the toxic (Bellows et al., 1990). Thus, this biological eect of sodium acetate on an osteogenic population is somewhat similar to that reported for other sodium salts. Although 10 mM sodium acetate did not signi®cantly increase cellular proliferation, it probably produced a greater number of osteoprogenitor cells from the mixed population. The conjecture is supported by the ®nding of a large number of small, discrete nodules produced by the cultures exposed to 10 mM sodium acetate. However, this enhancement had no biological advantage because net bone mass was not increased by exposure to 10 mM acetate. As 10 mM acetate had no eects on cell proliferation, its enhancement of mineralization following the removal of b-glycerophosphate suggests eects on the progression phase of the lifecycle of the osteoblast (Bellows et al., 1993). Sodium acetate at 20 mM severely inhibited cell proliferation and bone-nodule formation and mineralization, and could be considered toxic. As sodium acetate is widely used as a buer in total parenteral nutrition and renal dialysis, it may produce iatrogenic osteopenia when infused into patients. Our study suggests that this may occur not only by the enhancement of resorption (by secondary hyperparathyroidism), but also by the inhibition of matrix deposition due to inhibition of the proliferative phase of the osteoblast. A reduction of sodium acetate concentrations in these solutions may help prevent iatrogenic
L.A. Visconti et al. / Archives of Oral Biology 43 (1998) 729±733
induction of metabolic bone diseases. The dentist should be aware of these potential adverse eects of sodium acetate when evaluating oral bone loss in medically compromised patients. Acknowledgements This study was in partial ful®lment of the requirements for the Master of Science degree by Dr. Visconti and was supported by the Kidney Foundation of Canada (Manitoba Branch). We thank Delores Suga for technical assistance. References Alrey, A.C., Hegg, A., Craswell, P., 1980. Metabolism and toxicity of aluminum in renal failure. Am. J. Clin. Nutr. 33, 1509±1516. Bellows, C.G., Aubin, J.E., 1989. Determination of numbers in osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev. Biol. 132, 8±13. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., Antosz, M.E., 1986. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif. Tissue Int. 38, 143±154. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., 1987. Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvarial cells in vitro. Endocrinology 121, 1985±1992. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., 1991. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner. 14, 27±40. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., 1993. Dierential eects of ¯uoride during initiation and progression of mineralization of osteoid nodules formed in vitro. J. Bone Miner. Res. 8, 1357±1363. Bellows, C.G., Heersche, J.N.M., Aubin, J.E., 1990. The eects of ¯uoride on osteoblast progenitors in vitro. J. Bone Miner. Res. 5, S101±S105. Beresford, J.N., Gallagher, J.A., Poser, J.W., Russell, R.G.G., 1984. Production of osteocalcin by human bone cells in vitro. Eects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids. Metab. Bone Dis. Rel. Res. 5, 229±234. Ecarot-Charrier, B., Glorieux, F.H., van der Rest, M., Pereira, G., 1983. Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture. J. Cell Biol. 96, 639±643. Ellies, L.G., Heersche, J.N.M., Pruzanski, W., Vadas, P., Aubin, J.E., 1993. The role of phospholipase A2 in interleukin-1-a-mediated inhibition of mineralization of the osteoid formed by fetal rat calvaria cells in vitro. J. Dent. Res. 72, 18±24.
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Farley, J.R., Tarbaux, N.M., Vermeiden, J.P.W., Baylink, D.J., 1988. In vitro evidence that local and systemic eectors can regulate 3[H]-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory action(s) of embryonic chick bone extract. Calcif. Tissue Int. 42, 23±33. Gonzalez, F.M., Pearson, J.E., Garbus, S.B., Holbert, R.D., 1974. On the eects of acetate during hemodialysis. Trans. Amer. Soc. Artif. Int. Organs 20, 169±174. Gronowicz, G., Woodiel, F.N., McCarthy, M.B., Raisz, L.G., 1989. In vitro mineralization of fetal rat parietal bones in de®ned serum-free medium: eect of b-glycerol phosphate. J. Bone Miner. Res. 4, 313±324. Henderson, J.S., Johnson, R.B., 1995. The eects of smokeless tobacco extract on bone nodule formation and mineralization by chick osteoblasts in vitro. Archs Oral Biol. 40, 615±621. Hock, J.M., Centrella, M., Canalis, E., 1988. Insulin-like growth factor I has independent eects on bone matrix formation and cell replication. Endocrinology 122, 254±260. Johnson, R.B., Henderson, J.S., 1997. Enhancement by sodium orthovanadate of the formation and mineralization of bone nodules by chick osteoblasts in vitro. Archs. Oral Biol. 42, 271±276. Luna, L.G., 1968. Manual of Histologic Staining methods of the Armed Forces Institute of Pathology, 3rd Edition. McGraw-Hill, New York, pp. 176±177. Mansell, M.A., Wing, A.J., 1983. Acetate or bicarbonate for hemodialysis?. Br. Med. J. 287, 308±309. Mion, C.M., Hegstrom, R.M., Boen, S.T., Scribner, B.H., 1964. Substitution of sodium acetate for sodium bicarbonate in the bath ¯uid for hemodialysis. Trans. Am. Soc. Artif. Intern. Organs 10, 110±113. Novello, A., Kelsch, R.C., Easterling, R.E., 1976. Acetate intolerance during hemodialysis. Clin. Nephrol. 5, 29±32. Prior, J.C., Cameron, E.C.., Knickerbocker, W.J., Sweeny, V.P., Suchowersky, O., 1982. Dialysis encephalopathy and osteomalcic bone disease. Am. J. Med. 72, 33±42. Saitta, J.C., Lipkin, E.W., Howard, G.A., 1989. Acetate inhibition of chick bone cell proliferation and bone growth in vitro. J. Bone Miner. Res. 4, 379±386. Sherrard, D.J., Ott, S.M., Andress, D.L., 1985. Pseudohyperparathyroidism: syndrome associated with aluminum intoxication in patients with renal failure. Am. J. Med. 79, 127±130. Skutches, C.L., Holroyde, C.P., Myers, R.N., Paul, P., Reichard, G.A., 1979. Plasma acetate turnover and oxidation. J. Clin. Invest. 64, 708±713. Spindler, K.P., Shapiro, D.B., Gross, S.B., Brighton, C.T., Clark, C.C., 1989. The eect of ascorbic acid on the metabolism of rat calvarial bone cells in vitro. J. Orthop. Res. 7, 696±701. Veech, R.L., 1986. The toxic impact of parenteral solutions on the metabolism of cells: a hypothesis for physiological parenteral therapy. Am. J. Clin. Nutr. 44, 519±551.
PERGAMON
Archives of Oral Biology 43 (1998) 729±733
ORAL BIOLOGY
Eects of sodium acetate on rat bone-nodule formation and mineralization in vitro L.A. Visconti a, E.H.K. Yen a, b, R.B. Johnson c, * a
Department of Preventive Dental Science, University of Manitoba, Winnipeg, Manitoba, Canada Dean, University of British Columbia Faculty of Dentistry, Vancouver, British Columbia, Canada c Department of Diagnostic Sciences, University of Mississippi Medical Center, Jackson, MS 39216-4505, USA b
Accepted 20 February 1998
Abstract Sodium acetate reportedly promotes bone atrophy by inducing resorption and inhibiting osteoprogenitor-cell proliferation, but little is known about its eects on bone-matrix deposition and mineralization by a population containing osteoprogenitor cells. The objective here was to assess the eects of 1±20 mM sodium acetate on the proliferation and dierentiation of these cells and their resultant bone-nodule formation and mineralization in an in vitro assay. Exposure to 10 mM sodium acetate had no eect on cellular proliferation but signi®cantly increased the production and mineralization of bone nodules ( p < 0.01), suggesting that it aected osteoprogenitor dierentiation and subsequent metabolism. However, 10 mM acetate did not increase net bone mass. Dilutions of 1±5 and 20 mM inhibited cellular proliferation and resultant bone-nodule formation and mineralization, signi®cantly reducing the percentage bone area as compared to controls ( p < 0.001). These data suggest that 1±5 and 20 mM sodium acetate signi®cantly inhibit bone deposition, whereas 10 mM has no eects, which could contribute to iatrogenic metabolic bone disease in patients receiving either renal dialysis or total parenteral nutrition. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Sodium acetate; Bone matrix; Bone mineralization; Osteoblast; Osteoporosis
1. Introduction Patients on renal dialysis and those receiving total parenteral nutrition have a high probability of concurrently developing metabolic bone disease. Substitution of acetate for bicarbonate in haemodialysis ¯uids was proposed to simplify the procedure by eliminating the need for a separate pump for bicarbonate concentrate (Mion et al., 1964). Acetate (35 mM) is included in more than 90% of haemodialysis in the United States and United Kingdom (Veech, 1986). Toxic eects of acetate occur when it is converted to bicarbonate, which promotes the intracellular accumulation of cal-
Abbreviations: IGF-1, insulin-like growth factor-1, PBS, phosphate-buered saline, TCA, trichloroacetic acid. * Corresponding author.
cium, phosphate and pyrophosphate resulting in secondary hyperparathyroidism and eventual osteoporosis (Veech, 1986). In addition, the high concentrations of intracellular inorganic phosphate will chelate metals (such as aluminium) and exacerbate their cytotoxicity on brain and bone cells (Alrey et al., 1980; Prior et al., 1982; Sherrard et al., 1985). There has been little support for the idea of removing sodium acetate from these solutions, as it is a relatively inexpensive and eective buer and complications from its use are not considered signi®cant. Despite controversy over the relation between the serum acetate and metabolic bone disease, there are few studies on its eect on either the life-cycle or the metabolism of the osteoblast. In an in vitro study of chick osteoprogenitor cells, Saitta et al. (1989) reported that 5±20 mM sodium acetate decreased their rate of proliferation while increasing their alkaline phospha-
0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 8 ) 0 0 0 2 7 - 2
730
L.A. Visconti et al. / Archives of Oral Biology 43 (1998) 729±733
tase activity. However, that study was short and could not predict whether the resultant bone deposition or mineralization would be altered. There is no published information on the direct eects of sodium acetate on bone-matrix deposition and mineralization, although one in vitro study of fetal femurs showed that acetate decreased the dry weight of the bones, suggesting its use as a buer in solutions for total parenteral nutrition could promote bone atrophy (Saitta et al., 1989). Thus, it is worthwhile to investigate further the eects of acetate on the proliferation and metabolism of osteoprogenitor cells with the aim of acquiring additional information for designing preventive methods to minimize iatrogenic bone loss during renal dialysis or total parenteral nutrition. 2. Materials and methods 2.1. Explant procedure The procedure for the isolation of osteogenic cells was a modi®cation of two previously described techniques (Ecarot-Charrier et al., 1983; Beresford et al., 1984). Parietal bones were removed aseptically from 21-day-old embryos of female Sprague±Dawley rats. The periosteum was removed and the bones washed three times with PBS containing 1% antibiotics. Bones were then cut into fragments of 3±5 mm dia. and incubated in a collagenase solution [3 mg/ml type IA bacterial collagenase (from Clostridium histolyticum, collagen digestion activity = 550 units/mg; Sigma)] for 10 min at 378C to remove the ®broblast population. They were then washed with fetal bovine serum to inhibit enzymatic activity. Bone pieces (0.2±0.6 g) were placed in 60-mm tissue-culture dishes and incubated for 1 h at 378C to promote their adherence to the bottom of the dish. Growth medium (2 ml of a-minimal essential medium, pH 7.2, containing 2.2 g/l sodium bicarbonate + 15% fetal calf serum + 10% antibiotics; Sigma) was added to each dish and a further 2 ml of that medium added after 24 h. Growth medium was changed every second day thereafter. The cells reached con¯uence after 7 days. They were then released with trypsin±EDTA (1 ; Sigma), and plated in 35-mm tissue-culture dishes at a density of 2.5 104 cells/dish. 2.2. Cell isolation and growth Osteogenic cells were grown in vitro essentially by the techniques of Bellows et al. (1986): 3 104 cells were plated in 35-mm dishes and maintained in either standard medium [a-minimal essential medium supplemented with 1% antibiotics, 15% heat-inactivated fetal bovine serum (Sigma), 50 mg/ml ascorbic acid (Spindler et al., 1989), 10 mM sodium b-glyceropho-
sphate (Gronowicz et al., 1989) and 1 10ÿ7 M dexamethasone (Sigma) (Bellows et al., 1987)] or test medium, which was a modi®ed standard medium with 1, 3, 5, 10 or 20 mM sodium acetate substituted for equimolar amounts of sodium bicarbonate; this substitution does not diminish the buering capacity of the medium (Saitta et al., 1989). Some cultures were exposed to 10ÿ7 M human recombinant IGF-1 (Sigma) (Hock et al., 1988). The pH of the medium was adjusted to 7.4 before contact with the cells. Dishes were maintained at 378C in a humidi®ed atmosphere consisting of 95% air and 5% CO2. Medium was changed every 3 days. 2.3. Determination of cell proliferation The eect of sodium acetate on the proliferation of cells was determined by whole-cell counts and by evaluation of the incorporation of [3H] thymidine into DNA. During the ®nal 24 h of incubation, growth medium was replaced with serum-free medium. During the ®nal 2 h cells were exposed to [6ÿ3H] thymidine (1 mCi/ml spec. act., 29 Ci/mmol; Amersham Life Science). Medium was removed and cells were exposed to trypsin±EDTA (1 ; Sigma) for 5 min, the remaining adherent cells removed by scraping, and a 0.5-ml portion counted in a Coulter counter. A volume (1 ml) of the remaining cell suspension was centrifuged, the supernatant decanted, and the cells resuspended in 10% TCA for 20 min and centrifuged. This procedure was repeated. The TCA was decanted and the cells were solubilized in 0.5% sodium hydroxide for 60 min at 708C. The radioactivity was determined from a 100ml portion of the cell extract by liquid-scintillation analysis (Farley et al., 1988). Data were expressed as dis/min per 105 cells. 2.4. Bone-nodule formation The eects of sodium acetate on cell proliferation and resultant bone-matrix formation were determined in the nodule assay described by Bellows et al. (1986). Osteogenic cells were maintained for 21 days in medium supplemented with sodium acetate or 10ÿ7M IGF1, as previously described. At the end of the culture period, cells were ®xed with 10% neutral-buered formalin and bone nodules stained by the von Kossa method for calcium (Luna, 1968). Images of each culture were captured on computer disk and the nodules were counted and the percentage bone area calculated by densitometry using an image-analysis program [Microcomputer Imaging Device (MCID), Imaging Research; St. Catharines, Ontario]. Data were expressed as the number of bone nodules per culture and the percentage of the dish occupied by bone (percentage bone area).
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2.5. Determination of bone-nodule mineralization The eects of sodium acetate on bone-nodule mineralization were determined by measurement of 45Ca uptake into bone nodules, as described by Bellows et al. (1991) and Ellies et al. (1993). Cells were plated as previously described and were maintained for 18 days in standard medium (containing 0, 5, 10, 20 mM sodium acetate or 10ÿ7M IGF-1) without b-glycerophosphate, followed by a 3-day incubation in pulsechase medium [standard medium supplemented with 10 mM sodium b-glycerophosphate and 0.1 mCi 45Ca/ ml (spec. act. 50 mCi/mg Ca; Amersham)]. Phase-contrast photomicrographs were taken of each culture, images captured on a computer disk, and the percentage bone area measured by densitometry using the MCID image-analysis program. Following this incubation, cell layers were washed twice with PBS and exposed to 10% formic acid for 24 h. 45Ca radioactivity was assessed by liquid-scintillation analysis. Background counts were made from cultures that had been exposed to 45Ca, but not b-glycerophosphate, and were subtracted from counts in treatment groups in all experiments. Data were expressed as dis/min per mm2 to normalize 45Ca incorporation to nodule mass. 2.6. Statistical analysis Means were calculated for each group and number of nodules and dis/min compared by factorial analysis of variance and Shee comparisons. Percentages were compared by the Kruskall±Wallis non-parametric
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ANOVA and Dunn's multiple-comparison test. Dierences were considered signi®cant when p < 0.05.
3. Results All concentrations of sodium acetate promoted cell growth and mean cell numbers were higher in most cultures exposed to sodium acetate after day 1. At day 3 the incorporation of [3H] thymidine into DNA peaked in all groups except those with 5 and 10 mM acetate, which peaked after 5 days of incubation. Most concentrations of sodium acetate inhibited [3H] thymidine incorporation into DNA as compared to control or IGF-1 during days 3±7 of culture (Table 1). Wholecell counts at day 7 were signi®cantly lower in cultures exposed to 1±5 and 20 mM concentrations of sodium acetate; exposure to 10 mM produced no changes in cell numbers (Table 1). Sodium acetate at concentrations of 1±5 mM and 20 mM signi®cantly inhibited the percentage bone area, while 10 mM concentrations increased that area as compared to control or IGF-1 ( p < 0.001) (Table 2). However, no concentration of sodium acetate increased the percentage bone area compared to control values (Table 2). Sodium acetate at concentrations of 1±5 and 20 mM inhibited 45Ca incorporation into bone nodules ( p < 0.05); 10 mM sodium acetate enhanced radioisotope incorporation but was not signi®cantly more eective than IGF-1 (Table 3).
Table 1 Eects of various dilutions of sodium acetate (0±20 mM) and 10ÿ7M IGF-1 on [3H] thymidine incorporation (dis/min per 105 cells) into osteogenic cells 1±7 days after plating mM 0 1 3 5 10 20 IGF-1
Days 1
3 d
10.1620.81 (0.5120.08) 10.1420.87d (0.9320.13b,f) 9.442 0.32d (0.8320.09b) 4.922 0.84b,d (1.0620.14b,f) 3.582 0.29a,d (0.8620.12c) 0.352 0.03a,d (0.9220.12b,f) 26.4222.75a (0.6820.04)
5 d
53.672 3.34 (6.3720.83) 39.082 5.10c,d (6.0220.78) 28.422 13.35a,d (3.9920.56) 17.292 0.72a,d (2.7120.35b,e) 15.572 0.36a,d (6.8120.89) 0.8620.11a,d (0.9920.15a,d) 139.502 15.71a (4.6820.34)
Numbers in parentheses indicate cells 10ÿ5; n = 12 for each group. Signi®cantly dierent from O (control): ap < 0.001, bp < 0.01, cp < 0.05. Signi®cantly dierent from IGF-1: dp < 0.001, ep < 0.01, fp < 0.05.
7 d
16.1322.19 (10.3621.45) 1.9120.08a,d (7.4120.89) 1.5220.06a,d (4.5420.68b,e) 27.3620.30a,e (4.0120.61b,e) 45.9120.57 (8.9921.25) 0.3620.03a,d (1.3520.16a,d) 43.8425.61a (9.7820.93)
0.99 20.02 (12.822 2.27) 0.36 20.02a,f (7.302 0.87b,d) 0.29 20.05a,f (5.132 0.77b,d) 1.16 20.20 (8.072 1.29c,e) 0.56 20.11c (11.032 1.76) 0.2 20.03a,f (2.562 0.33a,d) 1.18 20.31 (15.492 2.01)
732
L.A. Visconti et al. / Archives of Oral Biology 43 (1998) 729±733
Table 2 Mean number of bone nodules and percentage bone area (2SEM) produced by cells exposed to various dilutions of sodium acetate or 10ÿ7M IGF-1 Bone Concentration (mM) nodules (No.) 0 (Control) 1 3 5 10 20 IGF-1
% area
e
32.2122.96f 14.1122.48a,d 12.6821.88a,d 15.6122.56b,d 21.0022.92c,e 4.1520.53a,d 48.8424.93a
31.4021.97 15.0021.54a,d 20.3021.58a,d 18.3621.14a,d 49.9223.42a,d 4.6421.28a,d 47.1025.21a
n = 12 for each group. Signi®cantly dierent from control: c p < 0.05. Signi®cantly dierent from IGF-1: f p < 0.05.
a
p < 0.001,
d
p < 0.001,
b
p < 0.01,
e
p < 0.01,
4. Discussion Sodium acetate is a compound found in many solutions for total parenteral nutrition and in some renaldialysis buers. It disrupts normal bone turnover by either producing secondary hyperparathyroidism (Veech, 1986) or inhibiting osteoprogenitor-cell proliferation (Saitta et al., 1989). To date, we believe only one study of the eects of sodium acetate on osteoprogenitor cells has been published (Saitta et al., 1989). That study was short (48 h) and reported the eects of the agent on the proliferation and not the progression phase of the life-cycle of the osteoblast. Long-term culture (15±21 days) of rat calvarial cells in the presence of ascorbic acid and sodium-b-glycerophosphate results in the formation of discrete, three-dimensional bone nodules. The number and size of these nodules can be quanti®ed and are subject to control by various agents (Henderson and Johnson, 1995; Johnson and Henderson, 1997). A nodule results from the proliferTable 3 45 Ca incorporation into bone nodules by cells exposed to various dilutions of sodium acetate or 10ÿ7M IGF-1 Concentration
Dis/min per mm2
0 1 3 5 10 20 IGF-1
3648.212412.42 1161.672127.78a,d 984.382147.75a,d 1143.982274.56a,d 6815.0721090.42b 336.43226.91a,d 4786.322622.18
n = 12 for each group. Signi®cantly dierent from control: ap < 0.001, bp < 0.01. Signi®cantly dierent from IGF-1: dp < 0.001.
ation and dierentiation of a discrete class of osteoprogenitor cells present at a low frequency in the population (Bellows and Aubin, 1989). Our study suggests that sodium acetate has adverse eects on the proliferation of that osteoprogenitor population and the deposition and mineralization of its matrix. Thus, it could potentially have adverse eects on both the deposition and resorption of bone in vivo. Renal-dialysis ¯uids contain 35±40 mM acetate (Saitta et al., 1989); ¯uids for total parenteral nutrition contain 48±146 mM acetate (Veech, 1986). Normal serum acetate is 0.01±0.4 mM (Skutches et al., 1979; Mansell and Wing, 1983) and many become routinely elevated to 3±5 mM after dialysis (Gonzalez et al., 1974; Novello et al., 1976; Mansell and Wing, 1983); concentrations exceeding 15 mM have been reported in patients on haemodialysis. Patients receiving total parenteral nutrition probably have a higher serum acetate because the solutions contain more acetate than in renal-dialysis ¯uids. Thus, the concentrations of acetate that we chose to test in vitro are similar to those in a medically compromised population. We found that 10 mM concentrations of sodium acetate increased bone-nodule formation and mineralization, whereas all other concentrations tested inhibited these functions. Agents such as sodium ¯uoride produce their maximal biological eects on osteogenic populations at concentrations close to the toxic (Bellows et al., 1990). Thus, this biological eect of sodium acetate on an osteogenic population is somewhat similar to that reported for other sodium salts. Although 10 mM sodium acetate did not signi®cantly increase cellular proliferation, it probably produced a greater number of osteoprogenitor cells from the mixed population. The conjecture is supported by the ®nding of a large number of small, discrete nodules produced by the cultures exposed to 10 mM sodium acetate. However, this enhancement had no biological advantage because net bone mass was not increased by exposure to 10 mM acetate. As 10 mM acetate had no eects on cell proliferation, its enhancement of mineralization following the removal of b-glycerophosphate suggests eects on the progression phase of the lifecycle of the osteoblast (Bellows et al., 1993). Sodium acetate at 20 mM severely inhibited cell proliferation and bone-nodule formation and mineralization, and could be considered toxic. As sodium acetate is widely used as a buer in total parenteral nutrition and renal dialysis, it may produce iatrogenic osteopenia when infused into patients. Our study suggests that this may occur not only by the enhancement of resorption (by secondary hyperparathyroidism), but also by the inhibition of matrix deposition due to inhibition of the proliferative phase of the osteoblast. A reduction of sodium acetate concentrations in these solutions may help prevent iatrogenic
L.A. Visconti et al. / Archives of Oral Biology 43 (1998) 729±733
induction of metabolic bone diseases. The dentist should be aware of these potential adverse eects of sodium acetate when evaluating oral bone loss in medically compromised patients. Acknowledgements This study was in partial ful®lment of the requirements for the Master of Science degree by Dr. Visconti and was supported by the Kidney Foundation of Canada (Manitoba Branch). We thank Delores Suga for technical assistance. References Alrey, A.C., Hegg, A., Craswell, P., 1980. Metabolism and toxicity of aluminum in renal failure. Am. J. Clin. Nutr. 33, 1509±1516. Bellows, C.G., Aubin, J.E., 1989. Determination of numbers in osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev. Biol. 132, 8±13. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., Antosz, M.E., 1986. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif. Tissue Int. 38, 143±154. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., 1987. Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvarial cells in vitro. Endocrinology 121, 1985±1992. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., 1991. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner. 14, 27±40. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., 1993. Dierential eects of ¯uoride during initiation and progression of mineralization of osteoid nodules formed in vitro. J. Bone Miner. Res. 8, 1357±1363. Bellows, C.G., Heersche, J.N.M., Aubin, J.E., 1990. The eects of ¯uoride on osteoblast progenitors in vitro. J. Bone Miner. Res. 5, S101±S105. Beresford, J.N., Gallagher, J.A., Poser, J.W., Russell, R.G.G., 1984. Production of osteocalcin by human bone cells in vitro. Eects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids. Metab. Bone Dis. Rel. Res. 5, 229±234. Ecarot-Charrier, B., Glorieux, F.H., van der Rest, M., Pereira, G., 1983. Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture. J. Cell Biol. 96, 639±643. Ellies, L.G., Heersche, J.N.M., Pruzanski, W., Vadas, P., Aubin, J.E., 1993. The role of phospholipase A2 in interleukin-1-a-mediated inhibition of mineralization of the osteoid formed by fetal rat calvaria cells in vitro. J. Dent. Res. 72, 18±24.
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Farley, J.R., Tarbaux, N.M., Vermeiden, J.P.W., Baylink, D.J., 1988. In vitro evidence that local and systemic eectors can regulate 3[H]-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory action(s) of embryonic chick bone extract. Calcif. Tissue Int. 42, 23±33. Gonzalez, F.M., Pearson, J.E., Garbus, S.B., Holbert, R.D., 1974. On the eects of acetate during hemodialysis. Trans. Amer. Soc. Artif. Int. Organs 20, 169±174. Gronowicz, G., Woodiel, F.N., McCarthy, M.B., Raisz, L.G., 1989. In vitro mineralization of fetal rat parietal bones in de®ned serum-free medium: eect of b-glycerol phosphate. J. Bone Miner. Res. 4, 313±324. Henderson, J.S., Johnson, R.B., 1995. The eects of smokeless tobacco extract on bone nodule formation and mineralization by chick osteoblasts in vitro. Archs Oral Biol. 40, 615±621. Hock, J.M., Centrella, M., Canalis, E., 1988. Insulin-like growth factor I has independent eects on bone matrix formation and cell replication. Endocrinology 122, 254±260. Johnson, R.B., Henderson, J.S., 1997. Enhancement by sodium orthovanadate of the formation and mineralization of bone nodules by chick osteoblasts in vitro. Archs. Oral Biol. 42, 271±276. Luna, L.G., 1968. Manual of Histologic Staining methods of the Armed Forces Institute of Pathology, 3rd Edition. McGraw-Hill, New York, pp. 176±177. Mansell, M.A., Wing, A.J., 1983. Acetate or bicarbonate for hemodialysis?. Br. Med. J. 287, 308±309. Mion, C.M., Hegstrom, R.M., Boen, S.T., Scribner, B.H., 1964. Substitution of sodium acetate for sodium bicarbonate in the bath ¯uid for hemodialysis. Trans. Am. Soc. Artif. Intern. Organs 10, 110±113. Novello, A., Kelsch, R.C., Easterling, R.E., 1976. Acetate intolerance during hemodialysis. Clin. Nephrol. 5, 29±32. Prior, J.C., Cameron, E.C.., Knickerbocker, W.J., Sweeny, V.P., Suchowersky, O., 1982. Dialysis encephalopathy and osteomalcic bone disease. Am. J. Med. 72, 33±42. Saitta, J.C., Lipkin, E.W., Howard, G.A., 1989. Acetate inhibition of chick bone cell proliferation and bone growth in vitro. J. Bone Miner. Res. 4, 379±386. Sherrard, D.J., Ott, S.M., Andress, D.L., 1985. Pseudohyperparathyroidism: syndrome associated with aluminum intoxication in patients with renal failure. Am. J. Med. 79, 127±130. Skutches, C.L., Holroyde, C.P., Myers, R.N., Paul, P., Reichard, G.A., 1979. Plasma acetate turnover and oxidation. J. Clin. Invest. 64, 708±713. Spindler, K.P., Shapiro, D.B., Gross, S.B., Brighton, C.T., Clark, C.C., 1989. The eect of ascorbic acid on the metabolism of rat calvarial bone cells in vitro. J. Orthop. Res. 7, 696±701. Veech, R.L., 1986. The toxic impact of parenteral solutions on the metabolism of cells: a hypothesis for physiological parenteral therapy. Am. J. Clin. Nutr. 44, 519±551.