A Tracer Study with Systemically and Locally Administered Dinitrophenylated Osteopontin
Laboratory for the Study of Calcified Tissues and Biomaterials, Faculty of Dentistry (AN,RMW,SFZ,MF) and Department of Pathology and Cell Biology, Faculty of Medicine (D-LG), Université de Montréal, Montreal, QC, Canada, and CIHR Group in Skeletal Development and Remodeling, School of Dentistry (HAG,GKH), University of Western Ontario, ON, Canada
Correspondence to: Antonio Nanci, Laboratory for the Study of Calcified Tissues and Biomaterials, Faculty of Dentistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada H3C 3J7. E-mail: antonio.nanci{at}umontreal.ca
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Summary |
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Key Words: tracer immunocytochemistry osteopontin albumin
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Introduction |
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Osteopontin is not only present in tissues but is also dissolved in serum and tissue fluids (Sodek et al. 2000; Rittling and Chambers 2004
). The potential contribution of these circulating forms of OPN to calcified tissue biology has received little attention. Indeed, a number of bone matrix components are normally found in the circulation, but these are generally regarded as metabolic byproducts of bone formation and resorption that have no function at distant sites. To our knowledge, there is only one study that has explored the fate of circulating OPN (VandenBos et al. 1999
). This light microscope study showed that intravenously administered [125I]OPN can be transported via the circulation and deposited in a number of calcified tissues. The amount of tracer administered was "three orders of magnitude" greater than the quantity of free OPN reported in human serum (concentrations in rat serum are not known). Under these conditions, some [125I]OPN was also found in enamel, a compartment in which the presence of OPN has not been revealed by biochemical assays and immunohistochemical techniques. Therefore, the authors concluded that they "could not exclude the possibility that the relatively high dose of injected OPN could have led to a somewhat artificial distribution pattern." These results nonetheless clearly highlighted the possibility that OPN in calcified tissues is not only derived from local cellular sources but may also be recruited from outside the local environment via the circulation.
Proteins have been tagged with chemical groups other than 125I to visualize them. One such alternative method is dinitrophenylation, involving the covalent addition of dinitrophenol (DNP) groups to the -lysine residues of proteins (Little and Eisen 1967
). This reaction, like iodination, generally does not alter the physiochemical properties of the tagged molecules (Kessler et al. 1982
). Thereafter, detection of tagged proteins is highly sensitive, because several DNP groups can be attached to a protein and the antigenicity of those groups is resistant to tissue processing conditions (Kessler et al. 1982
; Ghitescu and Bendayan 1992
). Dinitrophenylated albumin (ALB) has been administered to study vascular permeability (Ghitescu and Bendayan 1992
; Arshi et al. 2000
). In calcified tissues, DNP-tagged ALB was used to investigate the uptake of proteins by ameloblasts and odontoblasts (Nanci et al. 1996
). These cells, as well as osteoblasts, were shown to possess high levels of endocytotic activity and to take up protein non-selectively from the interstitial fluids.
The objective of the present study was to test the hypothesis that circulating forms of OPN may participate in bone formation. Tracer protocols such as those described above generally involve intravenous injections of relatively large amounts of proteins to saturate tissues throughout the body in quantities large enough to be detected. Such large dosages are rarely physiological. Our laboratory has developed an experimental system that allows the controlled administration of biological and chemical agents through a "surgical window" in the rat hemimandible (Vu et al. 1999; Orsini et al. 2001
). This system was used to infuse near-physiological amounts of DNP-tagged OPN and to demonstrate that the tracer molecules reach and are incorporated into the same sites at which endogenous OPN is believed to accumulate and act (Nanci 1999
).
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Materials and Methods |
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Surgical Procedures
Juvenile (56-week-old), male Wistar rats weighing 100 ± 10 g (Charles Rivers Canada; St-Constant, QC, Canada) were anesthetized with an intraperitoneal injection of a 1:1:2 mixture of Hypnorm (fentanyl citrate and fluanison; Janssen Pharmaceutica, Beerse, Belgium), Versed (midazolam; Hoffmann-LaRoche, Mississauga, ON, Canada), and distilled water. An 8-mm incision was made through the skin following an imaginary line extending between the auditory meatus and the lip commisure (Figure 1A). To expose the hemimandible, the masseter muscle was separated along the length of the fibers with a scalpel surgical blade (No 15C; Almedic, Montreal, QC, Canada). A dental drill fitted first with a size 010 carbide round burr (Brassler; Montreal, QC, Canada), followed by a size 014, was used to make a hole in the alveolar wall on the bony elevation associated with the apical end of the incisor at 2 mm from the posterior border of the ramus (Figure 1B). During drilling, the surgical site was irrigated with physiological saline. One- or 3-day Alzet osmotic minipumps [model 2001D for 1-day (8.0 µl/hr) and 1003D for 3-day (1.0 µl/hr); Alza, Palo Alto, CA] filled with complexes were slipped under the skin through a second incision made on the posterior region of the neck of the animal. A piece of vinyl tubing (size 0.72 x 1.22 mm; Scientific Commodities; Lake Havasu City, AZ) was hooked to the minipump and its free end passed through the neck area and underneath the masseter muscle. A metal catheter, made by bending a 20G1 needle (Becton-Dickinson; Rutherford, NJ), was used to connect the vinyl tubing to the bony hole. The metal catheter was immobilized against the bone surface with tissue adhesive (Indermil; Patterson Dental Supply, Montreal, QC, Canada) and bone cement (Zimmer; Warsaw, IN). The muscle was re-joined with 4-0 chromic gut sutures, and the skin was closed with 4-0 silk sutures (Patterson Dental Supply). The surgical site was cleaned and disinfected with 70% ethanol. The animals received an injection of Temgesic (buprenorphine HCl; Reckitt and Colman, Hull, UK) after surgery, and were fed with soft food containing Temgesic. X-rays, at 10 pulses/min, were taken to verify the positioning and the stability of the catheter (Figure 1C).
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The route of administration, concentrations, totals of amounts of complexes administered, and times of sacrifice of animals are summarized in Table 1. Negative control rats received only saline through the surgical window.
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Tissue Processing
The animals were anesthetized with 20% chloral hydrate solution (0.4 mg/g body weight; Fisher Scientific, Whitby, ON, Canada) and sacrificed by perfusion through the left ventricle with Ringer's lactate (Abbott Laboratories; Montreal, QC, Canada) for 30 sec, followed by a fixative solution consisting of 4% paraformaldehyde (BDH; Toronto, ON, Canada) and 0.1% glutaraldehyde (Electron Microscopy Sciences; Washington, PA) in 0.08 M sodium cacodylate (Electron Microscopy Sciences) buffer containing 0.05% calcium chloride (Sigma-Aldrich), pH 7.2, for 20 min. Treated and contralateral mandibles were taken, as well as the knees, and placed in the fixative solution for 24 hr at 4C. The hemimandibles and knees were washed with 0.1 M sodium cacodylate buffer, pH 7.2, and decalcified with 4.13% disodium ethylenediamine tetraacetic acid (Fisher Scientific) for 14 days at 4C (Warshawsky and Moore 1967). The decalcifying solution was changed every 2 days. Decalcified tissues were extensively washed in 0.1 M cacodylate buffer, pH 7.2, conventionally dehydrated in graded ethanols, and embedded in LR White resin (London Resin; Berkshire, UK) or osmicated with potassium ferrocyanide (Sigma-Aldrich)/reduced osmium tetroxide (Electron Microscopy Sciences) (Neiss 1984
), dehydrated in acetone, and embedded in Taab 812 epoxy resin (Marivac; Halifax, NS, Canada). Both resins were polymerized at 58C for 48 hr. Some samples were left calcified and similarly processed for embedding.
Light microscope observations were made on 1-µm semithin sections obtained with glass knives on a Reichert Jung Ultracut E ultramicrotome and stained with toluidine blue. Ultrathin sections 80100-nm thick were cut with a diamond knife and transferred on Formvar-coated (polyvinyl formate) 200-mesh nickel grids, and processed for postembedding colloidal gold immunolabeling.
Immunocytochemistry
Immunolocalization of proteins was done as previously described (Nanci et al. 1996) using the postembedding colloidal gold method (reviewed in Bendayan 1995
). Briefly, grid-mounted sections of osmicated tissues were first treated with a saturated aqueous solution of sodium metaperiodate (Fisher Scientific) (Bendayan and Zollinger 1983
). All sections were placed for 15 min on blocking solution consisting of 0.01 M PBS, pH 7.2, containing 1% PBS-ovalbumin (Sigma-Aldrich) and then transferred onto a drop of anti-DNP antibody (1:200, 1 hr; DAKO, Carpinteria, CA) to reveal the DNP-protein complexes, anti-OPN (1:10, 2 hr; LF-123, courtesy of Dr. L.W. Fisher, NIDCR, NIH, Bethesda, MD), or rabbit anti-rat ALB (1:60, 2 hr; ICN Pharmaceutical, Aurora, OH) antibodies to immunodetect endogenous molecules. Following incubation with primary antibodies, the grids were rinsed with PBS and placed again on the blocking solution for 15 min. A protein A-gold complex with particle size of 1012 nm (prepared in-house as described by Bendayan 1995
) was used to reveal the site of antibody binding. Finally, the grids were washed with PBS, followed by distilled water. All grids were stained with 4% aqueous uranyl acetate for 6 min and with lead citrate for 2 min and were examined in a JEOL JEM-1200 operated at 60 kV or a JEOL JEM-2011 transmission electron microscope operated at 80 kV.
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Results |
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Intravenous Injections of ALB-DNP
Endogenous ALB was immunodetected in the interstitial space between osteoblasts and in osteoid, but there was no significant accumulation in the bone matrix (Figure 2A). The presence of labeling between cells suggests that ALB can diffuse from the interstitial fluid, between cells and into osteoid. Tagged ALB was detected in the initial bone matrix deposited onto old bone (Figure 2B) and in osteoid (Figure 2C). Very few gold particles were found in association with cement lines, lamina limitans, or interfibrillar accumulations of non-collagenous matrix proteins. Calcified cartilage and bone exhibited almost no gold particles despite the occasional nearby presence of ALB-DNP in the tissue fluid (Figure 2D).
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Three-day Infusions of ALB-DNP
Despite infusion of amounts of ALB-DNP many-fold larger than those for OPN, this tagged protein accumulated mainly in the fibrin clot in the hole region. However, some gold particles were also found over the bone matrix near surfaces exposed by the drilling (Figure 5A). The labeling associated with bone diminished away from the bony hole, and no apparent tagged ALB was observed at the surface of calcified bone matrix. Tagged molecules diffused through osteocyte canaliculi, but these were not incorporated into the bone matrix (Figure 5B). Some ALB-DNP was found among the collagen fibrils of osteoid situated in proximity to the surgical window (Figure 5C).
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Discussion |
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Moreover, ultrastructural localizations of the tracer documented that exogenous OPN becomes incorporated into various compartments of bone in which endogenous OPN is believed to accumulate and act (Nanci 1999). One major advantage of the surgical window approach is that it allows the continuous administration of tracer in amounts that do not exceed the level of endogenous OPN constantly circulating through the tissues in the hemimandible.
ALB was employed as a control because this serum protein is found in bone but has a significantly lower inhibitory effect on hydroxyapatite formation than does OPN (Hunter et al. 1994). Some ALB-DNP is trapped in bone exposed during drilling, but the complexed protein, administered either systemically or locally, does not accumulate to any significant extent in non-collagenous matrix protein-enriched compartments. The relatively low affinity of this protein for bone is further demonstrated by the relatively modest labeling observed despite the fact that several-fold larger quantities of ALB-DNP were administered compared with OPN-DNP. Therefore, the behavior of ALB with respect to bone is not significantly changed by dinitrophenylation. This, together with the fact that OPN-DNP incorporates at sites in which endogenous molecules are believed to act, suggests that addition of DNP residues to a protein does not modify its affinity for bone.
Although it would be anticipated that circulating OPN would be attracted to sites of mineralization, this may not necessarily be the case. It has been suggested that circulating OPN is strongly bound to complement factor H and thus is sequestered, and that its activities are limited to their functional ranges (Fedarko et al. 2000). Our results suggest that in our experimental model, binding of OPN to factor H must occur over a time frame that allows the molecules to be available for incorporation into bone and/or that OPN prefers a calcifying matrix to complement factor H.
In conclusion, this first ultrastructural study demonstrates that dinitrophenylated OPN can be traced following either systemic or local administration and that the surgical window in the rat hemimandible is an efficient system for investigating the fate of proteins administered at low concentrations per unit time. It also clearly shows that circulating OPN can integrate into bone compartments such as mineralization foci and cement lines. This suggests that the action of this matrix protein extends beyond its microenvironment. Circulating molecules may have an important impact on initial events of bone formation, for which few molecules are generally required. The surgical window approach allows investigation of the fate of non-collagenous matrix proteins over time after they are released from the cells that manufacture them, and is applicable to a number of functional studies, such as determination of the behavior of different isoforms and evaluation of the activity of predicted functional groups.
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Acknowledgments |
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We thank Dr Charles E. Smith for his comments and discussions on the manuscript.
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Footnotes |
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Literature Cited |
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