The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack ... more The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin.
Monoclonal antibody HTP IV-#1 specifically recognizes a complexation-dependent neoepitope on bone... more Monoclonal antibody HTP IV-#1 specifically recognizes a complexation-dependent neoepitope on bone acidic glycoprotein-75 (BAG-75) and a Mr = 50 kDa fragment. Complexes of BAG-75 exist in situ, as shown by immunofluorescent staining of the primary spongiosa of rat tibial metaphysis and osteosarcoma cell micromass cultures with monoclonal antibody HTP IV-#1. Incorporation of BAG-75 into complexes by newborn growth plate and calvarial tissues was confirmed with a second, anti-BAG-75 peptide antibody (#503). Newly synthesized BAG-75 immunoprecipitated from mineralizing explant cultures of bone was present entirely in large macromolecular complexes, while immunoprecipitates from monolayer cultures of osteoblastic cells were previously shown to contain only monomeric Mr = 75 kDa BAG-75 and a 50 kDa fragment. Purified BAG-75 self-associated in vitro to form large spherical aggregate structures composed of a meshwork of 10 nm diameter fibrils. These structures have the capacity to sequester large amounts of phosphate ions as evidenced by X-ray microanalysis and by the fact that purified BAG-75 preparations, even after extensive dialysis against water, retained phosphate ions in concentrations more than 1,000-fold higher than can be accounted for by exchange calculations or by electrostatic binding. The ultrastructural distribution of immunogold-labeled BAG-75 in the primary spongiosa underlying the rat growth plate is distinct from that for other acidic phosphoproteins, osteopontin and bone sialoprotein. We conclude that BAG-75 self-associates in vitro and in vivo into microfibrillar complexes which are specifically recognized by monoclonal antibody HTP IV-#1. This propensity to self-associate into macromolecular complexes is not shared with acidic phosphoproteins osteopontin and bone sialoprotein. We hypothesize that an extracellular electronegative network of macromolecular BAG-75 complexes could serve an organizational role in forming bone or as a barrier restricting local diffusion of phosphate ions.
The goals of this study were to quantitate biochemical markers of bone metabolism on days 1-15 af... more The goals of this study were to quantitate biochemical markers of bone metabolism on days 1-15 after bilateral tibial marrow ablation surgery in young adult rats and to determine the effect of a single dose of methylprednisolone (2 mg/kg) or deflazacort (2.5 mg/kg) given at the time of ablation. Unexpectedly, serum calcium levels rose to a maximum of 15.9 mg/dl on day 7 after marrow ablation and remained above normal through day 15. This increase was blocked by a single intramedullary injection of methylprednisolone or deflazacort immediately following ablation; however, the fact that both drugs produced a characteristic rapid 3- to 10-fold increase in the serum alpha 2-macroglobulin level demonstrates that the drugs rapidly reached the circulation. Both methylprednisolone and deflazacort also inhibited intramedullary deposition of collagen by 40-60% on day 7, a time near which operated control animals achieved maximal accumulation of new bone in this model. Histological comparisons among the three experimental groups were largely consistent with biochemical results. The urinary hydroxyproline/creatine ratio for the operated control group doubled on day 3 and then returned to presurgical levels on day 7 and later. The timing and size of the hydroxyproline/creatinine peak, as well as the fact that the intratibial osteoclastic response peaks on days 8-10 after ablation, suggests it results from extratibial bone resorption induced by marrow ablation. Consistent with this rationale, urinary calcium excretion in operated controls rose 9-fold from day 0 to day 3 and appeared to plateau over the period from day 3 to day 9, before returning to a near presurgical level on day 15. Elevated excretion of calcium noted on days 9-15 in deflazacort-treated animals, which occurs in the absence of a detectable increase in resorption marker hydroxyproline, may however be due to the known action of glucocorticoids in increasing kidney filtration of calcium. In summary, this is the first report to show that bilateral tibial marrow ablation in rats causes a rapid hypercalcemia and calciuria which is accompanied initially by a peak of bone resorption marker urinary hydroxyproline. We speculate that the source of calcium and hydroxyproline is extratibial osteoclastic bone resorption induced by circulating cytokines whose release from ablated tibias or osteoclastogenic action is inhibitable by methylprednisolone and deflazacort.
Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during ext... more Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during extravasation from the vasculature, and upon attachment prior to resorption. Reverse transcriptase-PCR studies showed that two new alternatively spliced forms of chicken galectin-3, termed Gal-3TM1 and Gal-3TR1, were enriched and preferentially expressed in highly purified chicken osteoclast-like cells. Gal-3TM1 and Gal-3TR1 mRNA were also detected in chicken intestinal tissue, but not in kidney, liver, or lung. Gal-3TM1 and Gal-3TR1 messages both contain an open reading frame encoding a predicted 70-amino acid TM1 sequence inserted between the N-terminal Gly/Pro repeat domain and the carbohydrate recognition domain (exons 3 and 4). Gal-3TR1 mRNA contains an additional 241-bp sequence, which encodes a truncated open reading frame between the 4th and 5th exons, and, whose translation is expected to terminate within the carbohydrate recognition domain encompassing exons 4, 5, and 6. Immunoblotting and affinity chromatography showed that purified osteoclast preparations and intestinal homogenates contained a 36-kDa lactose-binding galectin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses on chymotryptic peptides from the 36-kDa lectin confirmed its identity as Gal-3TM1. The TM1 insert contains a single transmembrane-spanning region and a leucine zipper-like stalk domain that is predicted to position the intact carbohydrate recognition domain of Gal-3TM1 on the exterior surface of the plasma membrane. Immunofluorescent staining of chicken osteoclasts confirmed the expression of Gal-3TM1 at the plasma membrane. Gal-3TM1 is the first example of a galectin superfamily member capable of being expressed as a soluble protein and as a transmembrane protein.
Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity i... more Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity is rapidly and reproducibly filled by osteoblasts with new woven bone (days 6-8), which is then rapidly resorbed by osteoclasts (days 10-15). We showed previously (Magnuson et al., 1997) that marrow ablation induces a dramatic hypercalcemia and hypercalciuria in rats that unexpectedly peaked at the time of maximal osteogenesis and continued throughout the subsequent resorption phase. Based upon the amount of calcium mobilized and a peak of urinary hydroxyproline, we suggested that the hypercalcemia and hypercalciuria were due to increased systemic osteoclastic bone resorption induced by marrow ablation. We now apply a new enzyme-linked immunosorbent assay for rodent alpha(2)(I) N-telopeptide (NTx), a marker of bone resorption, to the marrow ablation model to demonstrate that excretion of NTx parallels that of calcium release in the operated control group. Specifically, maximal NTx/creatinine excretion coincides with the onset of hypercalcemia on days 7-8. A peak of NTx was also observed in methylprednisolone- and deflazacort-treated ablated animals. Analyses for urinary free deoxypyridinoline crosslink failed to detect a significant ablation-induced change in excretion. Interleukin 6 activity was increased in all operated control and glucocorticoid-treated groups after marrow ablation, whereas serum parathyroid hormone remained at presurgical levels in operated controls throughout the 15-day study period. The NTx results confirm that bilateral tibial marrow ablation induces a burst of extratibial bone resorption and hypercalcemia 7-8 days later. We have estimated that the osteogenic phase of the ablation model deposits 40 mg of calcium as hydroxyapatite crystals within the intramedullary cavity on days 6-8; this represents 33%-50% of the total blood calcium content of a young rat. We hypothesize that the size and rapidity of this demand for ionized calcium is met through an extratibial bone resorption pathway of osteoclast formation and activation that anticipates and fulfills this need, and that is initiated at the time of marrow ablation.
The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously repor... more The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously reported [Niggli, V., Penniston, J. T., & Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958], was freed of minor impurities by extensive washing while bound to the calmodulin-Sepharose column. The pure enzyme showed a single band of Mr 138000, which contained no stainable carbohydrate. The enzyme retained calmodulin-stimulable ATPase activity; with appropriate assay conditions, an activity of 21.2 mumol/(mg x min) was obtained. Amino acid analysis showed that the ATPase had a larger proportion of polar amino acids than do other integral membrane proteins. Despite this, the ATPase showed a tendency to form dimers and higher aggregates even in the presence of sodium dodecyl sulfate and urea. The enzyme required Mg2+ but showed little activity unless a second ion was added. With regard to this second ion, the enzyme responded to alkaline earth metal ions in the order Ca2+ greater than Sr2+ much greater than Ba2+. It was highly specific for ATP and was stimulated by Na+ or K+; in all of these properties it resembled the enzyme in unfractionated membranes. Limited proteolysis using trypsin yielded, at short times, many fragments of various molecular weights; continued proteolysis resulted in two trypsin-resistant fragments of Mr 81000 and 33500. Analysis of the time course of proteolysis indicated that the ATPase existed in two or more conformations that had differing susceptibilities to proteolysis. It is suggested that these correspond to active and inactive conformers of the enzyme.
Eruption is a highly localized process during which the bone resorption and formation that occur ... more Eruption is a highly localized process during which the bone resorption and formation that occur on opposite sides of the tooth are dependent upon the surrounding soft tissues, the true dental follicle externally and the enamel organ internally. To examine the ability of the enamel organ to cause eruption the external layer (dental follicle) was removed just prior to and up to 4 weeks before eruption in 13 mandibular premolars in dogs and eruption followed clinically, radiographically and histologically. None of the teeth without dental follicles erupted but three teeth from which the follicle was separated then replaced did erupt. These data indicate that the enamel organ without the dental follicle cannot support tooth eruption and provide indirect evidence for the central role of the dental follicle, alone or in combination with the enamel organ, in eruption.
Immunohistology of calvarial sections revealed that staining with monoclonal anti-osteopontin ant... more Immunohistology of calvarial sections revealed that staining with monoclonal anti-osteopontin antibodies (clone MPIIIB10) is minimal unless sections are first treated with EDTA. In contrast, following treatment of sections with EDTA, strong staining of mineralizing osteoid areas and osteoblast-like cells was noted (Fig. 1B). Immunostaining for osteopontin appeared to be specific in that controls which substituted rabbit IgG or normal mouse ascites fluid for monoclonal antibody, or which omitted monoclonal antibody uniformly gave background results (Fig. 1C). In an effort to circumvent problems of antibody accessibility we examined the immunoreactivity of OP when adsorbed to plastic and hydroxyapatite surfaces. Although OP bound to plastic surfaces is reactive with MPIIIB10 antibodies, OP adsorbed to hydroxyapatite crystal surfaces is not recognized by these antibodies as assessed by two detection methods. These results demonstrate that most or all of OP bound to hydroxyapatite exhibits a different conformation than when bound to plastic surfaces. On the basis of immunohistologic results with calvarial sections, we suggest that the conformation of native OP in bone and of isolated OP adsorbed to hydroxyapatite may be similar. Finally, solution circular dichroism and Fourier-transformed infrared spectroscopic studies indicate that the conformation of bone OP is dependent upon its concentration, and, secondarily to the presence or absence of calcium ion. With both spectroscopic methods, addition of calcium appeared to increase the extent of disordered structure. We suggest that these findings support our hypothesis that bone matrix proteins exhibit a different conformation when adsorbed on hydroxyapatite crystal surfaces. Assumption of a more organized secondary structure in concentrated OP solutions (i.e., 15 mg/ml) is consistent with these results in that local concentrations of OP within a semisolid matrix may approach or exceed levels used here.
The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack ... more The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin.
Monoclonal antibody HTP IV-#1 specifically recognizes a complexation-dependent neoepitope on bone... more Monoclonal antibody HTP IV-#1 specifically recognizes a complexation-dependent neoepitope on bone acidic glycoprotein-75 (BAG-75) and a Mr = 50 kDa fragment. Complexes of BAG-75 exist in situ, as shown by immunofluorescent staining of the primary spongiosa of rat tibial metaphysis and osteosarcoma cell micromass cultures with monoclonal antibody HTP IV-#1. Incorporation of BAG-75 into complexes by newborn growth plate and calvarial tissues was confirmed with a second, anti-BAG-75 peptide antibody (#503). Newly synthesized BAG-75 immunoprecipitated from mineralizing explant cultures of bone was present entirely in large macromolecular complexes, while immunoprecipitates from monolayer cultures of osteoblastic cells were previously shown to contain only monomeric Mr = 75 kDa BAG-75 and a 50 kDa fragment. Purified BAG-75 self-associated in vitro to form large spherical aggregate structures composed of a meshwork of 10 nm diameter fibrils. These structures have the capacity to sequester large amounts of phosphate ions as evidenced by X-ray microanalysis and by the fact that purified BAG-75 preparations, even after extensive dialysis against water, retained phosphate ions in concentrations more than 1,000-fold higher than can be accounted for by exchange calculations or by electrostatic binding. The ultrastructural distribution of immunogold-labeled BAG-75 in the primary spongiosa underlying the rat growth plate is distinct from that for other acidic phosphoproteins, osteopontin and bone sialoprotein. We conclude that BAG-75 self-associates in vitro and in vivo into microfibrillar complexes which are specifically recognized by monoclonal antibody HTP IV-#1. This propensity to self-associate into macromolecular complexes is not shared with acidic phosphoproteins osteopontin and bone sialoprotein. We hypothesize that an extracellular electronegative network of macromolecular BAG-75 complexes could serve an organizational role in forming bone or as a barrier restricting local diffusion of phosphate ions.
The goals of this study were to quantitate biochemical markers of bone metabolism on days 1-15 af... more The goals of this study were to quantitate biochemical markers of bone metabolism on days 1-15 after bilateral tibial marrow ablation surgery in young adult rats and to determine the effect of a single dose of methylprednisolone (2 mg/kg) or deflazacort (2.5 mg/kg) given at the time of ablation. Unexpectedly, serum calcium levels rose to a maximum of 15.9 mg/dl on day 7 after marrow ablation and remained above normal through day 15. This increase was blocked by a single intramedullary injection of methylprednisolone or deflazacort immediately following ablation; however, the fact that both drugs produced a characteristic rapid 3- to 10-fold increase in the serum alpha 2-macroglobulin level demonstrates that the drugs rapidly reached the circulation. Both methylprednisolone and deflazacort also inhibited intramedullary deposition of collagen by 40-60% on day 7, a time near which operated control animals achieved maximal accumulation of new bone in this model. Histological comparisons among the three experimental groups were largely consistent with biochemical results. The urinary hydroxyproline/creatine ratio for the operated control group doubled on day 3 and then returned to presurgical levels on day 7 and later. The timing and size of the hydroxyproline/creatinine peak, as well as the fact that the intratibial osteoclastic response peaks on days 8-10 after ablation, suggests it results from extratibial bone resorption induced by marrow ablation. Consistent with this rationale, urinary calcium excretion in operated controls rose 9-fold from day 0 to day 3 and appeared to plateau over the period from day 3 to day 9, before returning to a near presurgical level on day 15. Elevated excretion of calcium noted on days 9-15 in deflazacort-treated animals, which occurs in the absence of a detectable increase in resorption marker hydroxyproline, may however be due to the known action of glucocorticoids in increasing kidney filtration of calcium. In summary, this is the first report to show that bilateral tibial marrow ablation in rats causes a rapid hypercalcemia and calciuria which is accompanied initially by a peak of bone resorption marker urinary hydroxyproline. We speculate that the source of calcium and hydroxyproline is extratibial osteoclastic bone resorption induced by circulating cytokines whose release from ablated tibias or osteoclastogenic action is inhibitable by methylprednisolone and deflazacort.
Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during ext... more Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during extravasation from the vasculature, and upon attachment prior to resorption. Reverse transcriptase-PCR studies showed that two new alternatively spliced forms of chicken galectin-3, termed Gal-3TM1 and Gal-3TR1, were enriched and preferentially expressed in highly purified chicken osteoclast-like cells. Gal-3TM1 and Gal-3TR1 mRNA were also detected in chicken intestinal tissue, but not in kidney, liver, or lung. Gal-3TM1 and Gal-3TR1 messages both contain an open reading frame encoding a predicted 70-amino acid TM1 sequence inserted between the N-terminal Gly/Pro repeat domain and the carbohydrate recognition domain (exons 3 and 4). Gal-3TR1 mRNA contains an additional 241-bp sequence, which encodes a truncated open reading frame between the 4th and 5th exons, and, whose translation is expected to terminate within the carbohydrate recognition domain encompassing exons 4, 5, and 6. Immunoblotting and affinity chromatography showed that purified osteoclast preparations and intestinal homogenates contained a 36-kDa lactose-binding galectin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses on chymotryptic peptides from the 36-kDa lectin confirmed its identity as Gal-3TM1. The TM1 insert contains a single transmembrane-spanning region and a leucine zipper-like stalk domain that is predicted to position the intact carbohydrate recognition domain of Gal-3TM1 on the exterior surface of the plasma membrane. Immunofluorescent staining of chicken osteoclasts confirmed the expression of Gal-3TM1 at the plasma membrane. Gal-3TM1 is the first example of a galectin superfamily member capable of being expressed as a soluble protein and as a transmembrane protein.
Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity i... more Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity is rapidly and reproducibly filled by osteoblasts with new woven bone (days 6-8), which is then rapidly resorbed by osteoclasts (days 10-15). We showed previously (Magnuson et al., 1997) that marrow ablation induces a dramatic hypercalcemia and hypercalciuria in rats that unexpectedly peaked at the time of maximal osteogenesis and continued throughout the subsequent resorption phase. Based upon the amount of calcium mobilized and a peak of urinary hydroxyproline, we suggested that the hypercalcemia and hypercalciuria were due to increased systemic osteoclastic bone resorption induced by marrow ablation. We now apply a new enzyme-linked immunosorbent assay for rodent alpha(2)(I) N-telopeptide (NTx), a marker of bone resorption, to the marrow ablation model to demonstrate that excretion of NTx parallels that of calcium release in the operated control group. Specifically, maximal NTx/creatinine excretion coincides with the onset of hypercalcemia on days 7-8. A peak of NTx was also observed in methylprednisolone- and deflazacort-treated ablated animals. Analyses for urinary free deoxypyridinoline crosslink failed to detect a significant ablation-induced change in excretion. Interleukin 6 activity was increased in all operated control and glucocorticoid-treated groups after marrow ablation, whereas serum parathyroid hormone remained at presurgical levels in operated controls throughout the 15-day study period. The NTx results confirm that bilateral tibial marrow ablation induces a burst of extratibial bone resorption and hypercalcemia 7-8 days later. We have estimated that the osteogenic phase of the ablation model deposits 40 mg of calcium as hydroxyapatite crystals within the intramedullary cavity on days 6-8; this represents 33%-50% of the total blood calcium content of a young rat. We hypothesize that the size and rapidity of this demand for ionized calcium is met through an extratibial bone resorption pathway of osteoclast formation and activation that anticipates and fulfills this need, and that is initiated at the time of marrow ablation.
The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously repor... more The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously reported [Niggli, V., Penniston, J. T., & Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958], was freed of minor impurities by extensive washing while bound to the calmodulin-Sepharose column. The pure enzyme showed a single band of Mr 138000, which contained no stainable carbohydrate. The enzyme retained calmodulin-stimulable ATPase activity; with appropriate assay conditions, an activity of 21.2 mumol/(mg x min) was obtained. Amino acid analysis showed that the ATPase had a larger proportion of polar amino acids than do other integral membrane proteins. Despite this, the ATPase showed a tendency to form dimers and higher aggregates even in the presence of sodium dodecyl sulfate and urea. The enzyme required Mg2+ but showed little activity unless a second ion was added. With regard to this second ion, the enzyme responded to alkaline earth metal ions in the order Ca2+ greater than Sr2+ much greater than Ba2+. It was highly specific for ATP and was stimulated by Na+ or K+; in all of these properties it resembled the enzyme in unfractionated membranes. Limited proteolysis using trypsin yielded, at short times, many fragments of various molecular weights; continued proteolysis resulted in two trypsin-resistant fragments of Mr 81000 and 33500. Analysis of the time course of proteolysis indicated that the ATPase existed in two or more conformations that had differing susceptibilities to proteolysis. It is suggested that these correspond to active and inactive conformers of the enzyme.
Eruption is a highly localized process during which the bone resorption and formation that occur ... more Eruption is a highly localized process during which the bone resorption and formation that occur on opposite sides of the tooth are dependent upon the surrounding soft tissues, the true dental follicle externally and the enamel organ internally. To examine the ability of the enamel organ to cause eruption the external layer (dental follicle) was removed just prior to and up to 4 weeks before eruption in 13 mandibular premolars in dogs and eruption followed clinically, radiographically and histologically. None of the teeth without dental follicles erupted but three teeth from which the follicle was separated then replaced did erupt. These data indicate that the enamel organ without the dental follicle cannot support tooth eruption and provide indirect evidence for the central role of the dental follicle, alone or in combination with the enamel organ, in eruption.
Immunohistology of calvarial sections revealed that staining with monoclonal anti-osteopontin ant... more Immunohistology of calvarial sections revealed that staining with monoclonal anti-osteopontin antibodies (clone MPIIIB10) is minimal unless sections are first treated with EDTA. In contrast, following treatment of sections with EDTA, strong staining of mineralizing osteoid areas and osteoblast-like cells was noted (Fig. 1B). Immunostaining for osteopontin appeared to be specific in that controls which substituted rabbit IgG or normal mouse ascites fluid for monoclonal antibody, or which omitted monoclonal antibody uniformly gave background results (Fig. 1C). In an effort to circumvent problems of antibody accessibility we examined the immunoreactivity of OP when adsorbed to plastic and hydroxyapatite surfaces. Although OP bound to plastic surfaces is reactive with MPIIIB10 antibodies, OP adsorbed to hydroxyapatite crystal surfaces is not recognized by these antibodies as assessed by two detection methods. These results demonstrate that most or all of OP bound to hydroxyapatite exhibits a different conformation than when bound to plastic surfaces. On the basis of immunohistologic results with calvarial sections, we suggest that the conformation of native OP in bone and of isolated OP adsorbed to hydroxyapatite may be similar. Finally, solution circular dichroism and Fourier-transformed infrared spectroscopic studies indicate that the conformation of bone OP is dependent upon its concentration, and, secondarily to the presence or absence of calcium ion. With both spectroscopic methods, addition of calcium appeared to increase the extent of disordered structure. We suggest that these findings support our hypothesis that bone matrix proteins exhibit a different conformation when adsorbed on hydroxyapatite crystal surfaces. Assumption of a more organized secondary structure in concentrated OP solutions (i.e., 15 mg/ml) is consistent with these results in that local concentrations of OP within a semisolid matrix may approach or exceed levels used here.
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Papers by Jeffrey Gorski