Copyright ©The Histochemical Society, Inc.

A Tracer Study with Systemically and Locally Administered Dinitrophenylated Osteopontin

Antonio Nanci, Rima M. Wazen, Sylvia F. Zalzal, Micheline Fortin, Harvey A. Goldberg, Graeme K. Hunter and Dorin-Lucian Ghitescu

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


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Osteopontin (OPN), a major non-collagenous matrix protein of bone, is also found in tissue fluids and in the circulation. It is still not clear whether circulating OPN contributes to bone formation. To elucidate this question, rat OPN was tagged with dinitrophenol groups and administered to rats either intravenously or by infusion with an osmotic minipump through a "surgical window" in the bone of the hemimandible. Dinitrophenylated rat albumin (ALB) was used as a control. The presence and distribution of tagged proteins were revealed by immunogold labeling on sections of tibia and alveolar bone. Tagged molecules of OPN were found in mineralization foci, surfaces and interfaces, and matrix accumulations among calcified collagen fibrils. Even though dinitrophenylated ALB was administered at several-fold higher concentrations, it did not accumulate in these sites. These results show that circulating OPN can be incorporated into specific compartments of forming bone and suggest that such molecules may play a more important role than previously suspected. (J Histochem Cytochem 52:1591–1600, 2004)

Key Words: tracer • immunocytochemistry • osteopontin • albumin


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
OSTEOPONTIN (OPN) is an acidic, phosphorylated glycoprotein that is rich in aspartic acid, glutamic acid, and serine residues, and contains an RGD sequence for cell attachment (reviewed by Sodek et al. 2000Go). It is a member of the SIBLING family of proteins secreted by osteoblasts in forming bone (Fisher et al. 2001Go), where it accumulates in mineralization foci, in spaces between mineralized collagen fibrils, and in cement lines (Nanci 1999Go). In addition, it is expressed by a broad variety of cells (Denhardt et al. 2001Go; Qin et al. 2004Go), suggesting a multiplicity of functions in diverse biological events. Because it has a strong affinity for hydroxyapatite, it accumulates in calcified matrices, where it modulates formation and growth of mineral (Hunter et al. 1994Go). OPN expression increases as a result of injury and disease and is closely associated with ectopic calcified deposits (Steitz et al. 2002Go).

Osteopontin is not only present in tissues but is also dissolved in serum and tissue fluids (Sodek et al. 2000Go; Rittling and Chambers 2004Go). 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. 1999Go). 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 {varepsilon}-lysine residues of proteins (Little and Eisen 1967Go). This reaction, like iodination, generally does not alter the physiochemical properties of the tagged molecules (Kessler et al. 1982Go). 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. 1982Go; Ghitescu and Bendayan 1992Go). Dinitrophenylated albumin (ALB) has been administered to study vascular permeability (Ghitescu and Bendayan 1992Go; Arshi et al. 2000Go). In calcified tissues, DNP-tagged ALB was used to investigate the uptake of proteins by ameloblasts and odontoblasts (Nanci et al. 1996Go). 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. 1999Go; Orsini et al. 2001Go). 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 1999Go).


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Dinitrophenylation
Albumin (Sigma-Aldrich Canada; Oakville, ON, Canada) and bone-extracted rat OPN (Goldberg and Sodek 1994Go) were tagged with DNP according to the method described by Little and Eisen (1967)Go. Briefly, distilled water, potassium carbonate (BDH; Toronto, ON, Canada), 2,4-dinitrobenzene sulfonic acid (Sigma-Aldrich), and proteins were mixed in a 0.2:1:2:4 ratio by weight for 48 hr at room temperature, shielded from light. At the end of the reaction, the unreacted DNP was removed by dialysis for 72 hr against 0.01 M phosphate-buffered saline (PBS) containing 0.85% NaCl, pH 7.2, in 12 kD dialysis tubing (Sigma-Aldrich). The complexes were concentrated by centrifugation at 300–350 x g, 4C for 20 min, in Centricon YM-10 ultrafiltration tubes (Millipore; Bedford, MA).

Surgical Procedures
Juvenile (5–6-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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Figure 1

(A) Photograph illustrating the separated masseter muscle, the hole (arrows) drilled in the hemimandible, and the metal catheter linking the tubing of the osmotic minipump to the hole. (B) Isolated hemimandible with catheter in place. (C) X-ray showing the position of the catheter. (D–G) Light micrographs illustrating the histological appearance of a bone defect 1 day after its creation. The drilling site (DS) contains a granulation tissue with fibrin, bone debris, an inflammatory cell infiltrate, and multinucleated giant cells (MGC). Osteoclasts (Ocl) are frequently apposed to bone surfaces in proximity to the hole. (H,I) Light micrographs illustrating the histological appearance of the drilling site at day 3 after creation of the surgical window. Note that there is already new bone formation along the walls of the bony hole [osteoblast (Ob)]. A cement line (CL) demarcates the new from the old bone. The new bone is less metachromatic and contains plump cuboidal osteoblasts with well-developed Golgi regions (G, here appearing as pale paranuclear regions). Bars: A–C = 1 cm; D = 300 µm; E,F = 75 µm; G = 150 µm; H,I = 10 µm.

 
Six male Wistar rats weighing 100 ± 10 g (Charles River Canada) were anesthetized and injected through the jugular vein with DNP-tagged ALB or OPN.

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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Table 1

Experimental outline

 
All animal procedures and experimental protocols described above were in accordance with guidelines of the Comité de Déontologie de l'Expérimentation sur les Animaux of the Université de Montréal.

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 1967Go). 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 1984Go), 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 80–100-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. 1996Go) using the postembedding colloidal gold method (reviewed in Bendayan 1995Go). Briefly, grid-mounted sections of osmicated tissues were first treated with a saturated aqueous solution of sodium metaperiodate (Fisher Scientific) (Bendayan and Zollinger 1983Go). 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 10–12 nm (prepared in-house as described by Bendayan 1995Go) 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.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Histological Observations
Light microscopic observations of semithin sections in the region of the surgical window of animals infused with complexes for 1 day showed the characteristics of an early tissue repair reaction (Figure 1D). Bone debris produced during drilling (Figure 1F), fibrin clot (Figure 1E), and a cellular infiltrate comprising inflammatory cells, some osteoclasts and/or multinucleated giant cells, and fibroblast-like cells (Figures 1D–1G) were present at the periphery of the hole. Although osteoclasts were seen on bone debris and in the bone at the periphery, their presence was not visibly increased in animals infused with dinitrophenylated conjugates (Figures 1E and 1G) compared with controls infused with saline only (data not shown). As early as 3 days after the start of infusion, evidence of new bone formation was observed in animals infused either with saline only or with DNP-protein complexes (Figures 1H and 1I).

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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Figure 2

Electron micrographs illustrating the distribution of endogenous albumin (ALB) and of dinitrophenylated albumin (ALB-DNP) following intravenous injection. (A) Endogenous ALB is abundantly present in the intercellular space (arrows) between osteoblasts and in osteoid, but comparatively less in bone. (B–D) Similarly, tagged ALB is found in osteoid, but very few gold particles are associated with interfibrillar accumulations of non-collagenous matrix (arrowheads), cement lines (CL), or calcified bone matrix. RBC, red blood cell; Ob, osteoblast. Bars = 500 nm.

 
Intravenous Injections of OPN-DNP
At 24 hr following intravenous injections of OPN-DNP, tagged molecules were immunodetected on the surface of exposed calcified cartilage spicules in the primary spongiosa of the tibia (Figure 3A). In some mixed spicules, cement lines between calcified cartilage and bone or between adjacent layers of bone exhibited gold particles (Figure 3B). In general, bone was labeled along its surface but not in deeper regions, indicating that the unlabeled regions probably formed before the tracer was administered. Gold particles were found in mineralization foci in the osteoid seam and over accumulations of interfibrillar matrix in bone (Figure 3C). Endogenous OPN was immunodetected in these areas (Figure 3D).



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Figure 3

Immunocytochemical preparations of tibial bone (A–C) following a single intravenous injection of dinitrophenylated osteopontin (OPN-DNP) and (D) for endogenous osteopontin (OPN). (A) The lamina limitans (LL) coating calcified cartilage frequently incorporates tagged molecules. (B) These are also found in the cement line (CL) separating adjacent regions of bone in mixed spicules and (C) in interfibrillar matrix accumulations (arrowheads) among the calcified collagen. (D) Characteristic labeling for osteopontin over a CL and interfibrillar matrix accumulations. N, nucleus; Ob, osteoblast. Bars = 500 nm.

 
One-day Infusions of OPN-DNP
When high doses of OPN-DNP (Table 1) were infused over a 24-hr period, labeling was observed both at the drill site and at distant sites such as the growth plate. Gold particles were found in cement lines, interfibrillar matrix accumulations in bone (Figure 4A), and mineralization foci in osteoid (Figure 4B). At the drilling site, there was a concentration of tagged molecules on exposed bone surfaces, but many of them were also trapped in the adjacent fibrin clot (Figure 4C). Drilling caused microfractures and disrupted the collagen-packing network, resulting in diffusion of tracers into deeper bone (data not shown).



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Figure 4

Immunocytochemical preparations of (A,B) tibial and (C) alveolar bone after a 24-hr infusion of high (A–C) and near-physiological (D,E) doses of dinitrophenylated osteopontin (OPN-DNP). (A) Labeling was found on cement lines (CL) separating calcified cartilage from bone and, in some regions, over matrix accumulations (arrowheads) among the calcified collagen fibrils. (B) Dinitrophenylated OPN was occasionally associated with mineralization foci in osteoid. (C) The fibrin clot at the drill site sequesters some of the tagged molecules, particularly when high amounts of tracer are infused. When lower concentrations are administered, tracer accumulates mainly on bone surfaces exposed during drilling either (D) along the wall of the bony hole or (E) slightly below the surface, where adjacent layers of bone sometimes pull apart along cement lines (arrowheads). Such defects allow diffusion of tagged molecules to deeper regions of bone. Bars: A,B = 200 nm; C–E = 500 nm.

 
When near-physiological concentrations of OPN-DNP (Table 1) were infused for 24 hr, tagged proteins were found mainly in the surface layer of bone surrounding the hole and in the fibrin clot at the surgical site (Figure 4D). The density of labeling over these compartments appeared to be somewhat less than with the higher dose (compare Figures 4C and 4D). Some tracer molecules were found in deeper regions of bone along surfaces exposed during the surgical procedure, such as osteocyte canaliculi and split cement lines (Figure 4E). Very few gold particles were observed in deeper, undisturbed bone regions. Tagged molecules were not detected in tissue sections from tibia. With the tissue-processing and incubation conditions used in this study, no endogenous OPN was immunodetected in interstitial/circulating fluid compartments.

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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Figure 5

Immunocytochemical preparations of alveolar bone after a 3-day infusion of ALB-DNP. (A,C) Tagged ALB is mainly found in the fibrin clot along the walls of the bony hole and in osteoid near the drill sites. Note the absence of labeling on the exposed bone surface (arrowheads). (B) Some tracer diffuses along osteocyte canaliculi. Bars = 500 nm.

 
Three-day Infusions of OPN-DNP
Some mineralization foci (Figure 6A) were labeled with OPN-DNP, and the tagged molecules integrated into both osteoid and newly formed bone (Figure 6B). OPN-DNP was also detected near the drill site along surfaces destined for resorption by osteoclasts or around small bone debris surrounded by macrophages (Figure 6C). Like endogenous OPN (Figure 6E), infused OPN-DNP was present in the lamina limitans, surrounding osteocytes and coating the bone surface near bone lining cells (Figure 6D).



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Figure 6

Electron micrographs of alveolar bone after 3-day infusions of near-physiological doses of dinitrophenylated osteopontin (OPN-DNP) (A–D) immunolabeled for dinitrophenol and (E) endogenous osteopontin. (A) Tagged molecules become incorporated into new bone formed during the infusion interval at mineralization foci, (B) cement lines (CL), and (D) lamina limitans (LL) associated with bone lining cells (BLC). (C) They also coat bone debris at the drill site. (E) Endogenous osteopontin typically accumulates in cement lines between adjacent layers of bone and to a lesser extent in the lamina limitans surrounding osteocyte lacunae. Ob, osteoblast; Oc, osteocyte. Bars = 500 nm.

 
Controls
Only a few randomly distributed gold particles were observed over tissue sections from animals injected with saline and incubated with anti-DNP antibody (data not shown). No significant immunolabeling was seen in contralateral hemimandible and tibial tissues. Incubation of tissue sections with protein A-gold likewise resulted in little background labeling.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The results of this study demonstrate that OPN can be conjugated to DNP and that at least some OPN, transported via the blood circulation or diffusing locally in interstitial fluid, can integrate into bone under normal formative conditions or at sites of damage, a result consistent with the study of VandenBos et al. (1999)Go.

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 1999Go). 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. 1994Go). 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. 2000Go). 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.


    Acknowledgments
 
Supported by the Canadian Institutes of Health Research (CIHR).

We thank Dr Charles E. Smith for his comments and discussions on the manuscript.


    Footnotes
 
Received for publication June 21, 2004; accepted September 13, 2004


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 Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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