Connr<r i i ~ C / ~ ,3 S ( 1 4 1 , pp.

197-705 175 1-39]

Ti.ssiie R C J P ~ I I -Vol. 0 1996 OPA (Overwas Puhlisherh Association)
Reprints available dircctly from the publisher Amsterdam B V. Published in The Netherlands
Photocopying permitted hy license only under license hy Cordon and Breach Science Publishers
Printed in Malaysia

Osteopontin: An Interfacial Extracellular Matrix

Protein in Mineralized Tissues
MARC D. McKEE* and ANTONIO NANCI

Luborutor? fiw Electron Mirroscopy. Depurtment of Stomatolog?: Fuculh of Deiitistn, Uuiwrsitl dr Moritre!al. MontrPaI. Quebec, Canada
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(RrceiL'ed I Dec.enibei- 1995; Re\,ised 3 Januun 1996; Accepted 22 Murch 1996)

Among the noncollagenous matrix proteins found in mineralized tissues (MTs), col-
loidal-gold immunocytochemistry has demonstrated that the ultrastructural distribu-
tion of osteopontin (OPN) is unique in that this protein preferentially accumulates at MT
interfaces. In bone, OPN is present as a major component of cell- and matrix-matrix
interfacial structures termed laminae limitantes and cement lines, respectively. Here, we
review the implications of this distinct, interfacial tissue distribution as it relates to the
properties and functional motifs of OPN (e.g. RGD, polyAsp, phosphorylation) in differ-
ent MTs, and more specifically, how it pertains to current theory on the cellular and
extracellular matrix (ECM) events associated with bone remodeling. The production of
OPN as one of the earliest, and latest, secretory activities of the osteoblast lineage is dis-
For personal use only.

cussed, together with a consideration of the role of OPN in cement lines and laminae lim-
itantes in bone and in other normal, pathological and healing MTs such as teeth, kidney
stones, bone wound healing and implant osseointegration. Further to its ability to influ-
ence cell dynamics, calcification and possibly tissue cohesion in MTs, it is proposed that
OPN in cement lines may also promote adhesion between apposing substrata. With
regard to this latter function, it is suggested that the molecular interactions within, and
biomechanical properties of, such an OPN-rich interfacial zone may be important in
minimizing strain-induced fatigue damage and microcrack propagation in bone and
across other MT interfaces.

K e y o r d s : Osteogenesia, bone remodeling, cement line. macrophage, kidney stone, wound healing

INTRODUCTION constituent molecules. Many, if not most, of these pro-

In normal mineralized tissues (MTs) such as bones
and teeth, and at pathological calcification sites such
as kidney stones and atherosclerotic plaques, extrac-
tion of proteinaceous components from the extracellu-
lar organic matrices has revealed a plethora of
teins bind to the resident mineral and can be released
only after decalcification of the tissue. Prominent
among this class of mineral-binding, ECM molecules
is osteopontin (OPN), an acidic, phosphorylated sialo-
protein containing an Arg-Gly-Asp (RGD) cell adhe-
sion motif.".'] Recent investigations have documented

*Corresponding author. Tel.: 5 14 343-5763. Fax: 5 14 343-2233. E-mail: mckeem@ere.umontreal.ca

Address: Dentistry/Stomatology, Universite de Montreal, P. 0. Box 6128 - Station Centre Ville, Montreal. QC, Canada H3C 357.

197 [251]
 198 1252) M. D. McKEE and A. NANCI
the ability of OPN to regulate cell adhesion, spread-
ing, and migration in virro,['41 activities apparently
mediated in part by cell surface integrin receptors of
the a, family that are capable of binding various
~ ~ ] reported and sug-
RGD-containing l i g a n d ~ . Other
gested activities for OPN in MTs include regulation of
crystal maintenance of matrix/mineral
integrity (adhesion/cohesion),"ol and, as an integrin-
OPN complex, sensing of tissue strain (mechanosen-
sor).lIo1Binding of the integrin receptor(s) to its OPN
ligand induces signal transduction across the cell
membrane followed by a cascade of cytoskeletal
changes and other cytoplasmic events.["] Herein, we
review the distribution of OPN at various tissue and
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cell interfaces as determined by ultrastructural col-
loidal-gold immunocytochemistry, and present a dis-
cussion of the implications of site-specific
accumulation of this protein on cells, calcification and
matrix integrity in hard tissue formation, modeling,
remodeling and repair.
METHODOLOGY FOR LOCALIZING ECM
For personal use only.
MOLECULES IN MINERALIZED TISSUES
Unambiguous interpretation of immunocytochemical
data derived from studies on MTs requires an appreci-
ation of the nature of matrix-mineral relationships and
how they are affected by the various tissue processing
regimes commonly used to prepare biological speci-
mens for ultrastructural analysis.['21 Furthermore, the
intrinsic density of hard tissues, in which water occu-
pies a significantly lesser proportion of the tissue vol-
ume than in soft tissues, has traditionally been viewed
as a major obstacle in preventing rapid penetration of
fixative and complete fixation of internal biological
structure in MTs."'] Perhaps even more importantly,
the precise nature of tissue-specific, matrix-mineral
interactions might also potentially interfere with
achieving ideal tissue preservation and epitope expo-
sure. Therefore, depending on the questions being
asked, careful consideration must be given prior to the
selection and use of any tissue processing procedure
since the majority of these technical approaches are
potentially capable of inadvertently disturbing native
molecular relationships to such an extent that natural
tissue architecture is distorted by local translocation,
collapse a n d o r a more generalized loss of molecules
from the tissue. We have chosen to use the high-
resolution capabilities of electron microscopy.
together with the post-embedding colloidal-gold
immunocytochemical technique, to circumvent these
potential problems and to elucidate ECM composition
and organization in normal and pathological MTs. A
more complete discussion of this methodology as applied
to bone, cartilage and teeth is given elsewhere.["l
OSTEOPONTIN AND ECM
MINERALIZATION
OPN was first identified in bone ECM by biochemical
analysis of EDTA extracts."" Its presence in the min-
eral-binding compartment of bone led to its character-
ization as a phosphorylated organic moiety rich in
acidic amino acids and sialic acid, and a poly-Asp
sequence was subsequently identified. In vitro studies
demonstrated that native OPN inhibited both de n o w
apatite formation and crystal growth in a dose-
dependent manner,[6.71 whereas dephosphorylated
OPN greatly decreased these inhibitory effects.[71
Based on these data, it is likely that different phospho-
rylation states of this molecule may determine the
extent of its interaction with mineral in situ at different
locations within the MTs (see below). It is also likely
that the precise nature of its binding to mineral leads to
conformational changes that may actually participate
in orienting (exposing) other domains within OPN for
additional matrix or cell interactions. OPN has also
been identified in calcified cartilage, tooth dentin and
cementum, calcified atherosclerotic plaque and aortic
valves, and kidney stones (uropontin), and with regard
to the latter, has likewise been shown to inhibit cal-
cium oxalate formation.[*] Ultrastructural immunolo-
calization of OPN in both these normal and
pathological tissues has revealed an intimate associa-
tion of this protein with the organic crystal "ghosts"
surrounding individual c r y ~ t a l l i t e s . [ l ~Cr
~ 'ystal
~-~~~
ghosts are visualized by electron microscopy as an
electron-dense coating of organic material on the crys-
 OSTEOPONTIN IN MINERALIZED TISSUES
tals and are routinely observed at sites of calcification
within MTs.['~'Based on these and other observations,
it is likely that OPN controls crystal growth in vivo.
Studies are currently in progress to determine whether
OPN binding to mineral is nonspecific, or whether it is
specific and involves particular faces of the crystals.
OSTEOPONTIN AS A MEDIATOR OF CELL
DYNAMICS AT CELL-MATRIX INTERFACES
In most MTs, interfaces are numerous and can be
broadly classified as being either cell-matrix or
matrix-matrix interfaces.[l6]In bone, for example, the
former represents predominantly the interaction of the
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various bone cell lineages with surfaces of bone
matrix, and the latter represents the case where newer
bone is deposited onto older bone (or calcified carti-
lage in the case of endochondral ossification) to create
an interface between matrices made at distinctly dif-
ferent times and whose components are generally in
TABLE 1 OSTEOPONTIN ACCUMULATION AT TISSUE/SUBSTRATUM INTERFACES
For personal use only.
Interface
Bone - Bone
Bone - Calcified cartilage
Bone ~ Implant (Biomaterial)
Bone Culture substratum (in virro)
~ Polystyrene, glass and other culture materials.
Cementum - Dentin
Cementum ~ Cementum
CementudBone ~ LigamenUTendon
Renal calculi
Calcified atherosclerotic plaque
199 [253]
different orientations. As visualized by transmission
electron microscopy of decalcified sections, these
interfaces are usually characterized by the presence of
either an electron-dense, organic lamina limitans or a
cement line.['61Cement lines are found at matrix-
matrix interfaces where bone deposition has been pre-
ceded by a resorptive event (also called reversal lines),
or by a temporary cessation of matrix production (also
called resting lines). Laminae limitantes, on the other
hand, are generally somewhat thinner structures found
at bone (or cementum) surfaces interfacing with cells
(i.e. cell-matrix interfaces.) These sites include the
periphery of osteocyte (and cementocyte) lacunae and
bone (and cementum) canaliculi, and, quiescent bone
surfaces covered by bone-lining cells.
Particularly striking with regard to the distribution
of noncollagenous proteins in MTs in general is the
unique accumulation of OPN at various tissue inter-
faces including the cement lines (see Table I) and lam-
inae limitantes (see Table 11) found in a variety of
circumstances and in many different normal and
Tissue Structure or Substratum
ReversaKement lines (irregular contour; crenated).
RestingKement lines (smooth contour; non-crenated).
Edge of osseous defects (including after marrow ablation)
Surgically-created bone debris.
Concentric peri-osteocyte cement lines.
Mixed spicules of primary spongiosa (endochondral
ossification).
Osseointegrated titanium, hydroxyapatite and other
biomaterials.
Dentino-cementa1junction.
ReversaVCement lines (after natural root resorption).
Reparative cementum (after surgical root exposure).
RestingKement lines.
ReversaVCement lines (after natural root resorption)
Reparative cementum (after surgical root exposure).
Insertion sites, attachment /.ones (Sharpey's fibers)
Lamellae, striations, crystal ghosts
Cement lines / Laminae limitantes, mineralization front
(tidemark).
 M. D. McKEE and A. NANCl

TABLE I1 OSTEOPONTIN ACCUMULATION IN VlVO AT CELL-MATRIX (CALCIFIED) INTERFACES

Interface Location
Nr~rnicil

Preosteoblast - Bone / Substratum / Cartilage Resorbed and nonresorbed bone and cartilage surfaces
or other substrata such as biomaterials. Becomes
cement line.
Oateocyte Bone
- Osteocyte lacunae and cell process canaliculi.
Bone lining cell - Bone Quiescent bone surfaces
Osteoclast - Bone Initial stages ofthe bone remodeling hequence
Chondrocyte - Calcified cartilage Occa\ional chondrocyte lacunae.
Ceineiitoblast - Dentin Dentino-cementa1 junction formed during
cementogenesis. Becomes cement line.
Cementobla\t - Cementum Stratified cellular intrinsic fiber cementum. Become5
cement line (resting line). Resorbed root surfaces.
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Ccinentocyte - Cementum Ceinentocyte lacunae arid cell process canaliculi

Pcitliologrcrrl m d E.xperinierircr/

Macrophage-Calcified substratum Pathological or surgically exposed mineralized

surfaces (bone and tooth lesions/debris, calcified
atherosclerotic plaque). Phagocytosed particulates
Foreign body giant cells - Calcified substratum Implanted hydroxyapatite particles, bone and tooth
debris (after surgery).
Renal epithelium - Kidney stone Renal tubules of nephrolithic rats
Bone cells Osteopetrotic bone Multiple and thickened laminae limitantes at bone and
For personal use only.

cartilage surfaces.
Bone cells - Bone from 0060 src ’ ’knock-out‘ Laminae limitantes at bone surface.;

pathological tissues.i“’l For example, during the bone
remodeling sequence, OPN is deposited by cells of the
osteoblast lineage (preosteoblasts) to form a cement
(reversal) line (Fig. la) against a bone surface previ-
ously resorbed by osteoclasts. [Although osteoclasts
also produce OPN at certain times during the resorp
tive cycle, their participation in forming a discrete
structure (e.g. reversal line) within the ECM remains
uncertain, since resorption lacunae can be observed iri
~ 7 i v owithout evidence of OPN accumulation at the
resorbed surface.lfolMoreover, resting lines produced
by osteoblasts at sites not exposed to osteoclastic
resorption show structure and composition (OPN)
similar to reversal lines]. The phosphoserine (P-Ser)
content of cement lines can be visualized using a mon-
oclonal antibody against P-Ser (Fig. 1b), presumably
identifying a phosphorylated form of OPN. and possi-
bly other, as yet unidentified, phosphorylated organic
moieties at these sites. Subsequent secretory activity
by more mature, collagen-producing osteoblasts
results in the formation of bone matrix proper and the
accumulation of OPN throughout the mineralized
matrix compartment and in association with crystal
“ghosts” at small foci of calcification in the osteoid
(Fig. lc). Of particular note in osteoblasts is the
immunolabeling for OPN of Golgi compartments and
secretory granules known to contain procollagen fila-
ments (Fig. lc, inset), thus indicating that OPN can be
secreted in tandem with collagen and follows the same
secretory pathway in mature osteoblasts. Moreover,
gold particle labeling appears to be concentrated near
the electron-dense poles or at the center of cylindrical
 OSTEOPONTIN IN MINERALIZED TISSUES 201 [255]
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For personal use only.

FIGURE 1 Immunocytochemical localization of OPN in bone. (a) Cement lines (CL) in bone are planar accumulations of noncollagenous
organic matrix that label intensely for OPN and delineate a prior resorptive site (reversal line) or a site of temporary cessation of bone for-
mation (resting line). (b) Cement lines (CL) also immunolabel with anti-phosphoserine (P-Ser) monoclonal antibody, presumably reflecting
the phosphorylation of OPN at these sites. ( c ) During bone formation, OPN initially associates with the crystal “ghosts” demarcating early
foci of mineralization (arrows) among the collagen fibrils (Coll) of the osteoid. The inset illustrates an example of an OPN-labeled cylin-
drical distension of the Golgi apparatus known to contain procollagen filaments, thus demonstrating that OPN is secreted in tandem with
collagen and utilizes the same secretory pathway in mature osteoblasts. (d) When collagen production slows, and osteoblasts begin their
transition to bone-lining cells (Blc), immunogold labeling indicates that they produce an OPN-rich lamina limitans (1.L) at the bone surface.
Secretory granules in these cells, having a homogeneously dense content, frequently label intensely for OPN (inset). ( e ) Osteoclasts are fre-
quently observed directly apposed to the lamina limitans (LL) at bone surfaces. Regions of motile, adherent or resorptive osteoclasts found
against the lamina limitans include lamellipodia, the clearkealing zone (Ocl-CZ) or the ruffled border-each relationship depending criti-
cally on the resorptive status of the osteoclast. Bars equal 0.1 pm.

Golgi distensions and secretory granules. Near the end
of the bone formative phase, collagen production by
the osteoblast tapers off, and as the cells alter their
phenotype toward becoming bone-lining cells, they
produce a ‘coating’ of OPN that appears as a lamina
limitans at the bone surface (Fig. Id). During this tran-
sition, osteogenic cells frequently show secretory
granules with a more homogeneous content (perhaps
indicating an absence of collagen at this stage) that
label intensely for OPN (Fig. Id, inset). Ultimately, at
sites destined for resorption by osteoclasts, bone-
lining cells appear to retract, thus rendering the OPN-
coated bone surface available for interaction with
osteoclasts (Fig. le). This same sequence of histologi-
cal events is schematically illustrated in Figure 2, in
which OPN is a major component of the cement line
 202 12561 M. D. McKEE and A. NANCl

Stem cells Osteoclast

FIGURE 2 Schematic summary highlighting the interactions of bone cells with the ECM to form a cement line, then bone matrix proper.
and ultimately a lamina limitans at the completed bone surface. In this scenario, after matrix resorption by osteoclasts, the cement line and
lamina limitans are formed early and late, respectively, in the life cycle of the osteoblast lineage. Modified from McKee and Nanci.""'
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and lamina limitans. as well as the bone matrix proper.
A more complete description of OPN and the bone
remodeling sequence has been presented previ-
~ u s l y . [ Additional
~"~ interfacial sites of OPN accumu-
lation in bone include the lamina limitans delimiting
the wall of osteocyte lacunae, and that of the canaliculi
surrounding the network of cell processes coursing
throughout bone (Fig. 3a).
For personal use only.
tion and tightly adhere these cells to the adjacent ECM
via an integrin/RGD-mediated attachment mecha-
nism, and thus act as a mechanosensor for bone strain,
while at the same time possibly regulating mineral
dynamics that may be operative in such close proxim-
ity to the cell. In a similar manner, OPN in the lamina
limitans at the bone surface normally adjacent to bone-
lining cells may additionally act to 'prime' the surface

During the initial stages of wound repair in MTs,
macrophages are a major contributor of OPN that
binds as a lamina limitans to the exposed margins of
tissue defects and to the surfaces of MT debris parti-
cles (e.g. bone, enamel and dentin) found at the lesion
site (Figs. 3 b , ~ ) . ~In~ nephrolithic
'] rats (Fig. 3d), and
in humans, OPN (uropontin) is a major component of
the various lamellae, striations and crystal ghosts of
the organic matrix of calcium oxalate kidney
stones.'"] At least in rats, the contribution of OPN to
the urinary calculi appears to derive locally from sur-
rounding epithelial cells.
The presence of the cell adhesion RGD peptide in
OPN points to a role for this protein in promoting cell
attachment and spreading via integrin receptors.
Although most likely not involved in providing bio-
mechanical support and stability to bones, the OPN-
containing lamina limitans present at bone surfaces
subjacent to bone-lining cells, and around osteocytes
and their cell processes, may serve to accurately posi-
for future osteoclastic activity at these sites. Indeed,
our immunocytochemical data are consistent with the
notion that the RGD sequence of OPN exposed at
bone surfaces may be important initially for the inte-
grin-mediated formation of focal adhesionskontacts
of motile osteoclasts to the ECM of bone, then for
haptotaxis of these migratory cells, and then ulti-
mately for clear (sealing) zone fixation to the bone
surface, although this latter point remains controver-
sial. For tissue repair involving OPN production by
macrophages, OPN may serve as a macrophage adhe-
sion protein (as for OPN and osteoclasts) in that a
coating of OPN comprising the lamina limitans on
debris particles may act as a substratum for
macrophage attachment and spreading, and possibly
also as an opsonin, initiating signal transduction for
phagocytosis. In urolithiasis, it is likely that OPN
serves to inhibit calcium oxalate crystal growth, and
thus retard the progression, and possibly the aggrega-
tion, of renal calculi.
 OSTEOPONTIN IN MINERALIZED TISSUES 203 [257]
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For personal use only.

FIGURE 3 (a) OPN labeling over the lamina limitans (LL) delineating osteocyte lacunae and canaliculi, the latter shown in both longitudi-
nal and cross section (inset). In mineralized bone, OPN accumulates in irregular patches (P) of finely-reticulated material. (b) After bone
wounding by drilling, exposed bone surfaces (including particulate debris) are subjected to events comprising the inflammatory response and
ultimately tissue repair. Here, at 3 days post-drilling, macrophages (arrows) are found throughout the lesion and in close proximity to the
debris. Empty osteocyte lacunae are shown by arrowheads. (c) Macrophages in the lesions produce OPN, some of which binds to exposed
bone surfaces to form a lamina limitans (LL). (d) Kidney stone production in nephrolithic rats results in increased secretion of OPN by tubu-
lar epithelial cells. Labeling for OPN is associated with the various lamellae (asterisks) of the organic matrix in these renal caculi. Bars equal
(a), 0.2 pm; (b), 50 pm; (c), 0.2 pm; (d), 0.5 p m (inset, 0.2 pm).

OSTEOPONTIN AS A MEDIATOR OF TISSUE mation of covalent crosslinks.["] Recent evidence

ADHESIONKOHESION
ECM assembly in both hard and soft tissues frequently
involves initial noncovalent interactions (homophilic
and/or heterophilic) that are often followed by the for-
suggests that similar interactions may occur during the
integration of OPN into the ECM of MTs, and via this
or some other mechanism, OPN may participate in
maintaining tissue cohesionladhesion. Firstly, in addi-
tion to its mineral-binding properties, OPN has been
 204 125x1 M. D. McKEE and A. NANCI

shown to bind to osteocalcin and type I ~ o l l a g e n , ~ * ~ . ’ ~ ~ interactions in vitro: Inhibition of hydroxyapatite formation
both being prominent components of MT matrices.
Secondly, OPN is a substrate for the protein crosslink-
ing activity of tran~glutaminase,[~~~~’~
and this enzyme
has been shown to be capable of crosslinking OPN and
fibronectin.[*’] Thirdly, OPN is enriched at MT sites in
vivo that accommodate substantial tissue strain such as
the attachment zone (fibrocartilage and bone) of ten-
don and ligament osseous insertion sites (including the
periodontal ligament of the tooth, i.e. Sharpey’s
remodeled andor repaired bone and tooth
fibers),~’0~2s1
interfaces (e.g. cement lines),[’0*201 and the bone-
implant interface (i.e. after osse~integration).[*~~~~~ implants: Ultrastructural distribution and implications for min-
Thus, it is proposed that in addition to mediating cell
dynamics, calcification and possibly tissue cohesion
Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 10/04/13
and growth in a gelatin-gel. Bone Miner., 22, 147-159.
171 Hunter, G. K., Kyle, C. L. and Goldberg, H. A. (1994).
Modulation of crystal formation by bone phosphoproteins:
Structural specificity of the osteopontin-mediated inhibition of
hydroxyapatite formation, Biochern. J., 300,723-728.
[X] Worcester, E. M., Blumenthal, S. S.,Beshensky, A. M. and
Lewand, D. L. (1992). The calcium oxalate crystal growth
inhibitor protein produced by mouse kidney cortical cells in
culture is osteopontin, J. Bone Miner. Res., 7 , 1029-1036.
[9] Shiraga, H., Min, W., VanDusen, W. J., Clayman. M. D..
Miner, D., Terrell, C. H., Sherbotie, J. R., Foreman, J. W . ,
Przysiecki, C., Neilson, E. G. and Hoyer, J. R. (1992).
Inhibition of calcium oxalate crystal growth in vitro by uro-
pontin: Another member of the aspartic acid-rich protein
superfamily, Proc. Nutl. Acud. Sci. USA. 89,426-430.
101 McKee, M. D. and Nanci, A. (1996). Osteopontin at mineral-
ized tissue interfaces in bone. teeth and osseointegrated
eralized tissue formation, turnover and repair, Microsc. Res.
Tech., 33, 141-164.
111 Hruska, K. A,, Rolnick, F., Huskey, M., Alvarez, U . and

in MTs, OPN in cement lines may also act to promote
adhesion between apposing substrata, thereby main-
taining, for example, the overall integrity of bone dur-
ing the remodeling sequence, and the ‘bonding’ of
dissimilar tissues (or biocompatible materials)
together in biological composites such as teeth and
osseointegrated implants. Furthermore, the molecular
interactions within, and the biomechanical properties
of, an OPN-rich zone acting as a low-stiffness inter-
face may be important in minimizing strain-induced
For personal use only.
Cheresh, D. (1995). Engagement of the osteoclast integrin
a& by osteopontin stimulates phosphatidylinositol 3-
hydroxyl kinase activity, Endocrinology, 136, 2984-2992.
[I21 McKee, M. D. and Nanci, A. (1995). Postembedding col-
loidal-gold immunocytochemistry of noncollagenous extracel-
lular matrix proteins in mineralized tissues, M i crox. Res.
Tech., 31, 44-62.
[ 131 Bianco, P. (1990). Ultrastructural itnmunohistochemistry of
noncollagenous proteins in calcified tissues, In: U/tru.structurr
of SkeIetul Tissues: Bone und Curtiluge ir7 Health and
Disease, pp. 63-78. Ed. Bonucci, E. and Motta, P. M. Kluwer
Academic Publishers, Boston.
[I41 Franzen, A. and Heineglrd, D. (1985). Isolation and character-

ization of two sialoproteins present only in calcified bone.

fatigue damage and microcrack propagation in bone
[IS] Scherft, J. P. (1972). The lamina limitans of the organic matrix
and across other MT interfaces.
Acknowledgement
This work was supported by the Medical Research
Council of Canada and the FRSQ of Quebec.
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