ARTICLE |
Correspondence to: A. Nanci, Université de Montréal, Faculty of Dentistry/Stomatology, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada H3C 3J7. E-mail: nancia@ere.umontreal.ca
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphysealmetaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cellmatrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bonecalcified cartilage interface in some cases were thickened but generally did not appear affected at bonebone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption. (J Histochem Cytochem 48:10591077, 2000)
Key Words: rat, bone, hypocalcemia, hyperparathyroidism, osteomalacia, histomorphometry, TRAP, alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, cement line
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CALCIUM plays an important role in cellular physiology and homeostasis. It is stored when bone is deposited and liberated when it is resorbed. The serum calcium level is a major factor regulating bone remodeling (reviewed in
The aim of the present investigation was to clarify, using a combination of morphological approaches, the changes in bone status induced by chronic hypocalcemia in growing rats, an animal model frequently used to study the effects of metabolic factors on bone and bone cells (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypocalcemic Diet
Eighteen 21-day-old Wistar rats weighing about 45 g (Charles River; St-Constant, QC, Canada) were maintained on a cycle of 12-hr light/12-hr dark and were fed a completely calcium-free diet (Altromin DP1031; Rieper, Vandois, Italy) for 28 days. Another eight rats were used as controls and were given a normocalcemic diet (Altromin DP1000; Rieper) for the same period of time. The animals had free access to food and deionized water. Both the calcium-free and the normocalcic food contained 1000 IU of vitamin D/kg. An additional group of three rats was treated for 28 days with Altromin DP1031 containing 2500 IU of vitamin D/kg of food to rule out the possibility that the alterations observed reflected a vitamin D-dependent rickets. The experimental protocol was approved by the Comité de Déontologie de l'Expérimentation sur les Animaux of the Université de Montréal.
Blood Sampling and Tissue Processing for Histological Analyses
On Day 28, the rats were anesthetized with chloral hydrate (Sigma Chemical; St Louis, MO) and blood samples were drawn from the jugular vein for routine biochemical assays of (a) calcification parameters (calcium, phosphorus, alkaline phosphatase), (b) renal function (creatinine), (c) intestinal protein absorption (albumin), and (d) liver activity (aspartate aminotransferase and alanine transferase). The rats were then perfused through the left ventricle with lactated Ringer's solution (Abbott; Montreal, QC, Canada) for about 30 sec, followed by fixative for 20 min. The fixative solution consisted of either 4% paraformaldehyde + 0.1% glutaraldehyde in 0.08 M sodium cacodylate buffer, pH 7.3, or 1% glutaraldehyde in the same buffer. After perfusion, the tibiae were dissected, split longitudinally, and immersed in the corresponding fresh fixative solution for 3 hr (paraformaldehydeglutaraldehyde) or overnight (glutaraldehyde) at 4C. The proximal metaphysis of each hemitibia was then dissected and subdivided into small segments. These were processed for embedding in glycolmethacrylate (Merck/Schuchardt; Darmstadt, Germany) for enzyme histochemistry (
Histomorphometric Analysis
Histomorphometric analysis of undecalcified tibiae was carried out with an interactive image analyzer (IAS 2000; Delta Sistemi, Rome, Italy) on at least three ~2-µm-thick sections from each animal. The sections were cut at intervals of ~60 µm with a Reichert-Jung 1150/Autocut microtome. Structural variables (for nomenclature see
Cytochemical Staining
Thick sections for light microscopy were stained with azure IImethylene blue for routine examination or by the von Kossa method for calcium phosphate. Glycolmethacrylate sections were used to demonstrate alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities under the light microscope (
Immunolocalization of Noncollagenous Bone Matrix Proteins
Postembedding protein Acolloidal gold immunolabeling of tissue sections was applied to examine the presence and distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN) (reviewed in
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals from the present study (except for the hypocalcemic group supplemented with vitamin D) were also used to examine enamel formation in the mandibular incisors. Details of food and water consumption, and weight progression for the hypocalcemic and control groups are given in
Biochemical Assays
Results of the blood analyses are summarized in Table 1. The serum data showed conspicuous hypocalcemia in calcium-deprived rats (1.38 ± 0.09 vs 2.8 ± 0.10 mmol/liter; p<0.001), an increase in alkaline phosphatase and alanine aminotransferase, and normal values for phosphorus, creatinine, albumin, and aspartate aminotransferase.
|
Histomorphometry
The bone volume of calcium-deficient rats showed no significant difference from that of control rats (Table 2). Animals given a supplement of vitamin D (2500 IU of vitamin D) exhibited similar values. The absence of calcium in the diet resulted in a significant reduction of trabecular thickness, compared to the control group. The growth plate width was similar in three groups.
|
Both groups of rats given a calcium-free diet showed a significant increase in the osteoid parameters with respect to controls, as indicated by the values for osteoid volume, osteoid thickness, and osteoid surface (Table 3). Of note is that osteoid volume and thickness increased less in rats fed on a diet with 2500 IU/kg of vitamin D than in those maintained on a diet containing 1000 IU/kg. In both groups the osteoblast surface was also significantly greater than that in controls.
|
Osteoclast surface and osteoclast number were not significantly altered by hypocalcemia (Table 4). The eroded surface was significantly higher in treated rats receiving 1000 IU of vitamin D but not in those fed a diet with 2500 IU. However, in both these groups, almost all trabecular surfaces showed signs of bone remodeling.
|
Histology
The metaphyses of hypocalcemic rats appeared to consist of rather irregular trabeculae, delimiting medullary spaces that contained dilated capillary vessels and hemopoietic cells. Very few trabeculae were lined by typical bone lining cells, the majority of endosteal surfaces being covered by well-developed osteoblasts (Fig 1A and Fig 1B). Many osteoclasts were observed along the bone surfaces and mast cells were abundant near bone (Fig 1B, Fig 1D, and Fig 1E). Osteoclasts, particularly those near the growth plate, were often found apposed to unmineralized matrix (Fig 1D). The organization of osteoblasts along trabeculae varied according to the distance from the growth plate cartilage. Those at a distance generally formed a single row (Fig 1C and Fig 1E), whereas those nearer the growth plate appeared to be hyperplastic (Fig 1A). In both cases, the cells were rather large (mean diameter about 15 µm), and showed a deeply stained cytoplasm with a conspicuous Golgi apparatus and an eccentric nucleus with one or two nucleoli (Fig 1A and Fig 1E). Von Kossa staining confirmed that the central part of the trabeculae was calcified, whereas the peripheral bone matrix consisted of thick, uncalcified osteoid borders with a widened seam showing many mineralization foci (Fig 1C and Fig 1E).
|
Enzyme Histochemistry
In hypocalcemic rats, the majority of osteoclasts were TRAP-positive, and intensely reactive mononuclear cells were frequently seen around them (Fig 1F). TRAP activity was also present in osteoblasts and osteocytes (Fig 1F). The reaction product in these cells appeared as granular deposits which, in the case of osteoblasts, were aligned along the cell membrane facing osteoid (Fig 1F). Practically all the osteoblasts along the metaphyseal trabeculae showed TRAP staining, whereas those lining diaphyseal bone or the diaphyseal terminal portion of trabeculae were, in general, very weakly or not reactive. Osteoblasts also showed strong alkaline phosphatase activity. The reaction product was localized along their cell membrane (Fig 1G). In control animals, the majority of osteoblasts were TRAP-negative, reaction product being found only in some cells lining the initial portion of the trabeculae near calcifying cartilage, but all osteoblasts exhibited membrane-bound alkaline phosphatase (data not shown).
Electron Microscopy
The metaphyseal trabeculae of hypocalcemic rats consisted of a central calcified zone surrounded by a thick layer of osteoid showing abundant calcification foci (Fig 2). The trabeculae were lined by a variety of mononuclear cells (Fig 2). Many of them showed ultrastructural characteristics similar to those reported for active osteoblasts (
|
|
|
Osteoclasts in calcium-deficient rats showed either well-developed or incomplete ruffled borders and contacted either calcified and/or uncalcified bone matrix (Fig 5 Fig 6 Fig 7). They appeared to be less frequently apposed to bone surfaces showing a lamina limitans (Fig 5 Fig 6 Fig 7) (see
|
|
|
Ultrastructural Cytochemistry
Osteoclasts in hypocalcemic rats showed TRAP activity, even those in direct contact with osteoid tissue (Fig 8). Reaction product was localized in small electron-lucent vesicles in the region of the ruffled border and in larger lysosome-like profiles (Fig 8). Some reactivity was also found along the outer membrane of mitochondria (Fig 8).
|
Although many TRAP reaction product deposits were seen in osteoblasts by light microscopy, relatively few reactive granules were noted at the ultrastructural level, a situation probably due to the fact that the granules are small and are distributed throughout the large cell volume (Fig 9). However, systematic analysis of several serial sections revealed the presence of TRAP-positive vacuoles in the majority of osteoblasts from hypocalcemic rats.
|
Immunocytochemistry
Control rats showed a distribution for BSP, OC, and OPN similar to what has been previously reported for normal animals (data not shown;
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we applied a combination of morphological approaches to examine the effects of diet-induced chronic hypocalcemia in young, growing rats. Calcium deficiency in these animals produced severe bone alterations characterized by an increase in osteoid but no change in thickness of the growth plate. These data are consistent with some previous reports indicating that hypocalcemia induces calcification defects similar to those observed in rickets and osteomalacia (-hydroxylase activity in the kidney and a concomitant increase in 1,25(OH)2D3 (
There are few reports in the literature in which the effects of hypocalcemia were corroborated by histomorphometric measurements, and these generally show a reduction in trabecular bone volume, especially in adult animals (
All three noncollagenous bone matrix proteins examined were immunodetected in hypocalcemic rats. There were no major changes in their pattern of distribution or concentration at labeled sites that could be inferred from qualitative observation. Immunocytochemically, the most conspicuous difference in labeling between hypocalcemic and control rats was the presence of many mineralization foci, intensely immunoreactive for BSP, OC, and OPN, in the thickened osteoid. Despite the abundance of matrix vesicles, the presence of these foci indicates that initiation of mineralization must have occurred; however, its progression was clearly hampered. The abundance of osteoid tissue and the intense immunolabeling for noncollagenous matrix proteins in mineralization foci further suggest that the reduced availability of calcium, rather than an incompetent organic matrix, is the major factor for the alteration in mineralization observed in hypocalcemic rats.
The bone changes observed appear to be independent of abnormalities of kidney, liver, or intestine, at least so far as can be inferred from the normal levels of albumin and creatinine. Total plasma alkaline phosphatase levels were increased, suggesting an increase in bone formation. This value includes both bone and liver alkaline phosphatase. However, liver damage can be excluded because, despite the significant increase in alanine aminotransferase level in hypocalcemic rats, the aspartate aminotransferase/alanine aminotransferase ratio in these animals still remained greater than 1.
The presence of abundant rough endoplasmic reticulum, a conspicuous Golgi apparatus, and alkaline phosphatase activity along the membrane of many mononuclear cells lining the osteoid surface indicates that these cells belong to the osteoblast lineage. However, detection of TRAP reactivity in many of these lining cells suggests that they are also actively involved in degradative functions. TRAP-positive osteoblast-like cells have also been found in normal metaphyseal bone (
The inconsistent presence of a lamina limitans at sites where osteoclasts were apposed to the bone surface in hypocalcemic rats suggests that these cells are not obligatorily dependent on this interfacial structure for adherence to bone surfaces and for matrix degradation. In this context, it has been shown that osteoclasts can bind to native Type I collagen via 2ß1 integrin and denatured collagen via
vß3 integrin (
Another distinctive feature was that cement lines at the bonecalcified cartilage interface sometimes appeared to be thicker than those of control rats. These interfacial lines are believed to derive mainly from the differential deposition of matrix constituents by osteoblasts at the beginning and end of the bone-forming cycle (discussed in
The intracellular organization of osteoclasts in hypocalcemic rats resembled that of controls. However, they often showed incomplete or poorly formed ruffled borders. The occasional presence of crystallites within the membrane infoldings of the ruffled border in both hypocalcemic and control rats is perplexing, considering the acid pH at this site. Most osteoclasts in calcium-deficient animals exhibited widespread TRAP activity, even when located close to osteoid, suggesting that they can degrade uncalcified bone matrix. Osteoclast-mediated degradation of uncalcified collagen fibrils has also been reported in severe primary hyperparathyroidism (
TRAP activity was also detected in osteocytes, especially those located near osteoclasts, as previously reported in cases of hyperparathyroidism (
In conclusion, a calcium-free diet administered to young, growing rats reduced the serum calcium level to about half of normal values but did not significantly change bone volume. Osteoid volume, surface, and thickness increased. There were many mineralization foci in the widened osteoid seam, suggesting aborted attempts at mineralization. These were conspicuously immunoreactive for BSP, OC, and OPN. Bone turnover was increased and osteoblasts were hyperplastic. Most osteoblasts showed TRAP and alkaline phosphatase activity, thus resembling the BMU intermediate cells. Osteoclasts were all intensely stained for TRAP, showed poorly developed ruffled borders, and appeared to digest both calcified and uncalcified bone matrix. Although the alterations observed must take into consideration the effects of both calcium deficiency and growth, taken together the data suggest that severe hypocalcemia in young rats induces an osteomalacia-like state which, as is often the case, is characterized by bone changes caused by compensatory secondary hyperparathyroidism.
![]() |
Acknowledgments |
---|
Supported by grants from the Medical Research Council of Canada and the Ministry of University and Scientific and Technological Research (MURST) of Italy. P. Mocetti was the recipient of a fellowship from the Québec Ministry of Education.
We are grateful to Dr L.W. Fisher (National Institutes of Health; Bethesda, MD) for supplying the LF-87 BSP antibody, to Dr P.V. Hauschka (Harvard University; Boston, MA) for the OC antibody, to Dr R. Lepage (Hôpital St-Luc, Montreal, QC) for the biochemical analyses of blood samples, and to M. Fortin for diligent technical and photographic assistance.
Received for publication October 30, 1999; accepted March 15, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baron R, Vignery A, Horowitz M (1984) Lymphocytes, macrophages and the regulation of bone remodeling. In Peck WA, ed. Bone and Mineral Research. Annual 2. Amsterdam, Elsevier, 175-243
Bendayan M (1995) Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 29:1-159[Medline]
Bendayan M, Nanci A, Kan FWK (1987) Effect of tissue processing on colloidal gold cytochemistry. J Histochem Cytochem 35:983-996[Abstract]
Bianco P, Ballanti P, Bonucci E (1988) Tartrate-resistant acid phosphatase activity in rat osteoblasts and osteocytes. Calcif Tissue Int 43:167-171[Medline]
Bianco P, Bonucci E (1991) Endosteal surfaces in hyperparathyroidism: an enzyme cytochemical study on low-temperature-processed, glycol-methacrylate-embedded bone biopsies. Virchows Arch 419:425-431. [A]
Bianco P, Ponzi A, Bonucci E (1984) Basic and "special" stains for plastic sections in bone marrow histopathology, with special reference to May-Grunwald Giemsa and enzyme histochemistry. Basic Appl Histochem 28:265-279[Medline]
Bianco P, Riminucci M, Silvestrini G, Bonucci E, Termine JD, Fisher LW, Gehron Robey P (1993) Localization of bone sialoprotein (BSP) to Golgi and post-Golgi secretory structures in osteoblasts and to discrete sites in early bone matrix. J Histochem Cytochem 41:193-203
Bloom MA, Domm LV, Nalbandov AV, Bloom W (1958) Medullary bone of laying chickens. Am J Anat 102:411-453[Medline]
Bonucci E (1990) The ultrastructure of the osteocyte. In Bonucci E, Motta PM, eds. Ultrastructure of Skeletal Tissues. Boston, Kluwer Academic Publishers, 223-237
Bonucci E, Lo Cascio V, Adami S, Cominacini L, Galvanini G, Scuro A (1978) The ultrastructure of bone cells and bone matrix in human primary hyperparathyroidism. Virchows Arch 379:11-23. [A]
Bonucci E, Silvestrini G, Bianco P (1992) Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method. Histochemistry 97:323-327[Medline]
Clark SA, Ambrose WW, Anderson TR, Terrell RS, Toverud SU (1989) Ultrastructural localization of tartrate-resistant, purple acid phosphatase in rat osteoclasts by histochemistry and immunocytochemistry. J Bone Miner Res 4:399-405[Medline]
de Bernard B, Stagni N, Camerotto R, Vittur F, Zanetti M, Zambonin Zallone A, Teti A (1980) Influence of calcium depletion on medullary bone of laying hens. Calcif Tissue Int 32:221-228[Medline]
Dempster DW (1992) Bone remodeling. In Coe FL, Favus MJ, eds. Disorders of Bone and Mineral Metabolism. New York, Raven Press, 355-380
de Winter FR, Steendijk R (1975) The effect of a low-calcium diet in lactating rats: observations on the rapid development and repair of osteoporosis. Calcif Tissue Res 17:303-316[Medline]
Everts V, Beertsen W (1987) The role of microtubules in the phagocytosis of collagen by fibroblasts. Coll Relat Res 7:1-15[Medline]
Frens G (1973) Controlled nucleation for the regulation of particle size in monodispersed gold suspensions. Nature 241:20-22. [Phys Sci]
Hauschka PV, Frenkel J, DeMuth R, Gundberg CM (1983) Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid-containing proteins in developing bone. J Biol Chem 258:176-182
Helfrich MH, Nesbitt SA, Lakkakorpi PT, Barnes MJ, Bodary SC, Shankar G, Mason WT, Mendrick DL, Väänänen HK, Horton MA (1996) ß1 Integrins and osteoclast function: involvement in collagen recognition and bone resorption. Bone 19:317-328[Medline]
Jowsey J, GershonCohen J (1964) Effect of dietary calcium levels on production and reversal of experimental osteoporosis in cats. Proc Soc Exp Biol Med 116:437-441
Katsunuma N (1997) Molecular mechanisms of bone collagen degradation in bone resorption. J Bone Miner Metab 15:1-8
Liu C-C, Baylink DJ (1984) Differential response in alveolar bone osteoclasts residing at two different bone sites. Calcif Tissue Int 36:182-188[Medline]
Liu C-C, Rader JL, Gruber H, Baylink DJ (1982) Acute reduction in osteoclast number during bone repletion. Metab Bone Dis Relat Res 4:201-209[Medline]
McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A (1993) Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and 2HS-glycoprotein) proteins in rat bone. J Bone Miner Res 8:485-496[Medline]
McKee MD, Nanci A (1995) Postembedding colloidal-gold immunocytochemistry of noncollagenous proteins in mineralized tissues. Microsc Res Tech 31:44-62[Medline]
McKee MD, Nanci A (1996) Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microsc Res Tech 33:141-164[Medline]
Midura RJ, McQuillan DJ, Benham KJ, Fisher LW, Hascall VC (1990) A rat osteogenic cell line (UMR 106-01) synthesizes a highly sulfated form of bone sialoprotein. J Biol Chem 265:5285-5291
Muir JM, Hirsh J, Weitz JI, Andrew M, Young E, Shaughnessy SG (1997) A histomorphometric comparison of the effects of heparin and low-molecular-weight heparin on cancellous bone in rats. Blood 89:3236-3242
Nanci A (1999) Content and distribution of noncollagenous matrix proteins in bone and cementum: relationship to speed of formation and collagen packing density. J Struct Biol 126:256-269[Medline]
Nanci A, Ahluwalia JP, Zalzal S, Smith CE (1989) Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor. J Histochem Cytochem 37:1619-1633[Abstract]
Nanci A, McCarthy GF, Zalzal S, Clokie CML, Warshawsky H, McKee MD (1994) Tissue response to titanium implants in the rat tibia: ultrastructural, immunocytochemical and lectin-cyto-chemical characterization of the bone-titanium interface. Cells Mater 4:1-30
Nanci A, Mocetti P, Sakamoto Y, Kunikata M, Lozupone E, Bonucci E (2000a) Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor. J Histochem Cytochem 48:1043-1057
Nanci A, Zalzal S, Fortin M, Mangano C, Goldberg HA (2000b) Incorporation of circulating bone matrix proteins by implanted hydroxyapatite and at bone surfaces: implications for cement line formation and structuring of biomaterials. In Davies JE, ed. Bone Engineering. Toronto, em2, Inc. (In Press)
Nanci A, Zalzal S, Gotoh Y, McKee MD (1996) Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary culture. Microsc Res Tech 33:214-231[Medline]
Neiss WF (1984) Electron staining of the cell surface coat by osmium-low ferrocyanide. Histochemistry 80:231-242[Medline]
Nesbitt SA, Horton MA (1997) Trafficking of matrix collagens through bone-resorbing osteoclasts. Science 276:266-269
Ohya K (1994) Rats fed with a low-calcium diet as an in vivo experimental model for bone resorption. In Ogura H, ed. Pharmacological Approach to the Study of the Formation and the Resorption Mechanism of Hard Tissues. Tokyo, Ishiyaku Euroamerica, 93-113
Ornoy A, Wolinsky I, Guggenheim K (1974) Structure of long bones of rats and mice fed a low calcium diet. Calcif Tissue Res 15:71-76[Medline]
Parfitt AM (1990) Osteomalacia and related disorders. In Avioli LV, Krane SM, eds. Metabolic Bone Disease and Clinically Related Disorders. 2nd ed Philadelphia, WB Saunders, 329-396
Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR (1987) Bone histomorphometry: standardization of nomenclature, symbols, and units. J Bone Miner Res 2:595-610[Medline]
Persson P, GagnemoPersson R, Håkanson R (1993) The effect of high or low dietary calcium on bone and calcium homeostasis in young male rats. Calcif Tissue Int 52:460-464[Medline]
Pettifor JM, Marie PJ, Sly MR, du Bruyn DB, Ross F, Isdale JM, de Klerk WA, van der Walt WH (1984) The effect of differing dietary calcium and phosphorus contents on mineral metabolism and bone histomorphometry in young vitamin D-replete baboons. Calcif Tissue Int 36:668-676[Medline]
Riminucci M, Silvestrini G, Bonucci E, Fisher LW, Gerhon Robey P, Bianco P (1995) The anatomy of bone sialoprotein immunoreactive sites in bone as revealed by combined ultrastructural histochemistry and immunohistochemistry. Calcif Tissue Int 57:277-284[Medline]
Robinson JM, Karnovsky MJ (1983) Ultrastructural localization of several phosphatases with cerium. J Histochem Cytochem 31:1197-1208[Abstract]
Salomon CD (1972) Osteoporosis following calcium deficiency in rats. Calcif Tissue Res 8:320-333[Medline]
Salomon CD, Volpin G (1970) Fine structure of bone resorption in experimental osteoporosis caused by calcium deficient diet in rats. An electron microscopic study of compact bone. Calcif Tissue Res 4:80-82
Scherft JP, Groot CG (1990) The electron microscopic structure of the osteoblast. In Bonucci E, Motta PM, eds. Ultrastructure of Skeletal Tissues. Boston, Kluwer Academic Publishers, 209-222
Shen V, Birchman R, Xu R, Lindsay R, Dempster DW (1995) Short-term changes in histomorphometric and biochemical turnover markers and bone mineral density in estrogen- and/or dietary calcium-deficient rats. Bone 16:149-156[Medline]
Silberstein R, Melnick M, Greenberg G, Minkin C (1991) Bone remodeling in W/WV mast cell deficient mice. Bone 12:227-236[Medline]
Sissons HA, Kelman GJ, Marotti G (1984) Mechanisms of bone resorption in calcium-deficient rats. Calcif Tissue Int 36:711-721[Medline]
Stauffer M, Baylink D, Wergedal J, Rich C (1972) Bone repletion in calcium deficient rats fed a high calcium diet. Calcif Tissue Int 9:163-172
Stauffer M, Baylink D, Wergedal J, Rich C (1973) Decreased bone formation, mineralization, and enhanced resorption in calcium-deficient rats. Am J Physiol 225:269-276[Medline]
Thomas ML, Simmons DJ, Kidder L, Ibarra MJ (1991) Calcium metabolism and bone mineralization in female rats fed diets marginally sufficient in calcium: effects of increased dietary calcium intake. Bone Miner 12:1-14[Medline]
Thompson ER, Baylink DJ, Wergedal JE (1975) Increases in number and size of osteoclasts in response to calcium or phosphorus deficiency in the rat. Endocrinology 97:283-289[Abstract]
Väänänen HK (1996) Osteoclast function: biology and mechanisms. In Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of Bone Biology. San Diego, Academic Press, 103-114
Van den Bos T, Bronckers ALJJ, Goldberg HA, Beertsen W (1999) Blood circulation as a source of osteopontin in acellular extrinsic fiber cementum and other mineralizing tissues. J Dent Res 78:1688-1695[Abstract]
Warshawsky H, Moore G (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J Histochem Cytochem 15:542-549[Medline]
Weinreb M, Rodan GA, Thompson DD (1991) Immobilization-related bone loss in the rat is increased by calcium deficiency. Calcif Tissue Int 48:93-100[Medline]
Wergedal JE, Baylink DJ (1969) Distribution of acid and alkaline phosphatase activity in undemineralized sections of the rat tibial diaphysis. J Histochem Cytochem 17:799-806[Medline]
Yamamoto T, Yamagata A, Nagai H (1996) A histochemical study of tartrate-resistant acid phosphatase activity in rat osteoblasts. Acta Histochem Cytochem 29:221-225