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The ultrastructural distribution of two noncollagenous proteins, osteopontin (OPN) and osteocalcin (OC), originally extracted from bone matrix and proposed to play an important role in bone formation, was examined in the matrices of bone and cartilage from embryonic and postnatal chicken tibial growth plates by high-resolution immunocytochemistry using the colloidal gold technique. In bone, immunolabeling patterns using polyclonal antibodies against chicken OPN and OC were generally similar in that both showed an intense, but regionally variable, labeling of mineralized bone matrix and small mineralization loci dispersed throughout the osteoid and containing prominent condensed organic material. Unmineralized osteoid showed weak-to-moderate labeling. In the mineralized bone matrix proper, labeling was predominantly associated with amorphous, electron-dense patches of organic material among the collagen fibrils. In growth plate cartilage, both proteins first appeared related to calcified cartilage in the hypertrophic zone, although the labeling patterns were somewhat different. For OPN, gold particles were mostly associated with an organic lamina limitans-like density containing condensed, filamentous organic matrix at the periphery of small nodules and large masses of calcified cartilage, with additional moderate labeling throughout the interior of the calcified cartilage. For OC, labeling was observed over filamentous structures throughout the calcified cartilage matrix, with some, but less, labeling at the periphery. In the lowermost zones of the growth plate, the major reaction using both antibodies was found over a layer of dense, amorphous organic material at the periphery of the calcified cartilage at the future bone/calcified cartilage interface, a labeling pattern that persisted following bone deposition at these sites. OPN and to a lesser extent OC were also concentrated in cement (resting, reversal) lines. Throughout the bone and cartilage of the tibia, cells of both the osteoblastic and the osteoclastic lineages were found directly apposed to labeled surfaces and lamina limitans of organic matrix containing OPN and OC. In summary, it is concluded from the immunocytochemical data presented here that the association of OPN and OC with mineralized regions of the extracellular matrices of bone and cartilage and the accumulation of these proteins at tissue surfaces and interfaces are consistent with the hypotheses that they play a role in the extracellular mineralization process per se and/or that they may mediate cell adhesion and dynamics.
Anat Rec 1992 Dec
PMID:High-resolution immunolocalization of osteopontin and osteocalcin in bone and cartilage during endochondral ossification in the chicken tibia. 145 51

The cartilagenous tissue of mandibular condyles of newborn mice contains progenitor cells as well as young and mature chondrogenic cells. During in vitro cultivation of the tissue, progenitor cells undergo osteogenic differentiation and form new bone (Silbermann, M., D. Lewinson, H. Gonen, M. A. Lizarbe, and K. von der Mark. 1983. Anat. Rec. 206:373-383). We have studied the expression of genes that typify osteogenic differentiation in mandibular condyles during in vitro cultivation. RNAs of the genes for collagen type I, osteonectin, alkaline phosphatase, and bone gla protein were sequentially expressed in progenitor cells and hypertrophic chondrocytes during culture. Osteopontin expression peaked in both the early and the late phase of the differentiation process. The data indicate a distinct sequence of expression of osteoblast-specific genes during osteogenic differentiation and new bone formation in mandibular condyles.
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PMID:Gene expression during osteogenic differentiation in mandibular condyles in vitro. 169 Nov 90

Osteopontin (OPN), a noncollagenous, extracellular matrix sialoprotein found at relatively high levels in both normal and pathological mineralized tissues, is expressed by tissue-specific cells in bone, calcified cartilage, and teeth. On the other hand, a hallmark of OPN expression in pathologically mineralizing tissue, and in other soft tissues experiencing a more generalized type of necrotic injury, is the production of OPN by macrophages at the lesion site. In the present study, we have localized OPN and other noncollagenous proteins by ultrastructural colloidal-gold immunocytochemistry using a rat model in which mineralized tissue defects are surgically created in mandibular bone and teeth. The healing response was examined by immunocytochemistry and transmission electron microscopy at 10 min, 3 days and 7 days post-surgery using antibodies against OPN, bone sialoprotein, osteocalcin, bone acidic glycoprotein-75, fibronectin, and amelogenin. Whereas most of these proteins were characteristically distributed within their respective extracellular matrices as described previously, OPN was additionally observed to accumulate as a lamina limitans at surgically exposed bone and tooth surfaces, as well as at the surface of particulate, mineralized tissue debris. Intracellular labeling of the Golgi apparatus and secretory granules of macrophages at the lesion site demonstrated that OPN production by macrophages was a prominent secretory event of the inflammatory response during wound healing in mineralized tissues. Pseudopodal and lamellipodal cytoplasmic extensions of macrophages were observed in direct contact with the OPN-containing lamina limitans at these surfaces. Particulate, calcified debris internalized by macrophages also displayed a prominent surface "coating" of OPN. In conclusion, our interpretation of the present data is that OPN secreted by macrophages may serve as a macrophage adhesion protein, and where concentrated at the surface of small particulate, mineralized tissue debris, may act as an opsonin, thereby facilitating cell adhesion and phagocytosis by macrophages, a process likely mediated by integrin-binding, signal transduction, and cytoskeletal restructuring.
Anat Rec 1996 Jun
PMID:Secretion of Osteopontin by macrophages and its accumulation at tissue surfaces during wound healing in mineralized tissues: a potential requirement for macrophage adhesion and phagocytosis. 876 75

Enzymatic digestion of bone tissue potentially releases a mixture of precursor, differentiating, and mature cells. Conceptually, early fetal osteogenic tissue should provide a more uniform population of cells than late embryonic or newborn bone in which cells have already differentiated. In this context, we have applied sequential enzymatic digestion to obtain and culture cells from 15-16-day fetal rat cranial tissue, a developmental age where deposition of bone matrix has not yet started at this site. These cultures were compared with those of osteogenic cells isolated from newborn rat calvariae and grown under similar conditions. Matrix production and composition were examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The plated cells formed mineralized nodules by day 14. The presence of mineral was determined by von Kossa staining and backscattered electron imaging (BEI), and the accumulation of calcium and phosphorus within the nodules was demonstrated by X-ray microanalysis and elemental mapping. At early time intervals, cells were generally cuboidal in shape and showed a well-developed Golgi apparatus, which occasionally was immunoreactive for OPN. Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery, of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface. Osteocalcin immunoreactivity was also associated with electron-dense portions of the bone-like matrix. These data demonstrate the potential of presumptive fetal rat calvarial cells to form a bone-like matrix in vitro and suggest that the assembly and mineralization pattern show similarities to the process of intramembranous ossification. Such a culture system is of interest not only for studying cellular and matrix events of bone formation, but also factors which influence mesenchymal cells in committing themselves to the osteogenic pathway.
Anat Rec 1998 12
PMID:Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue. 984 6

Regeneration of damaged periodontal tissues is mediated by periodontal cells, but a major sub-population comprises highly differentiated cells that do not renew. To overcome the loss of specialized cell types caused by disease, various therapeutic approaches including cell transplants have been developed to promote cell re-population in periodontal tissues. As previous transplantation studies used unlabeled cells, that are indistinguishable from host cells, it has been difficult to assess the contributions of transplanted cells to the healing processes. To track the fate and differentiation of rat periodontal cells transplanted into periodontal wounds, we used collagen-coated fluorescent beads as a permanent endocytosed marker, or cells constitutively expressing beta-galactosidase. We assessed osteogenic cell differentiation with immunohistochemical staining for osteopontin and bone sialoprotein. Cells were transplanted into periodontal wounds created in Sprague--Dawley male rats that are null for beta-galactosidase. Defects were allowed to heal spontaneously (controls), or were closed with collagen implants mixed with beta-galactosidase-positive (Lac-Z) periodontal cells, or closed with collagen implants mixed with periodontal cells loaded with fluorescent beads. Animals were killed at 1 and 2 weeks after surgery and tissues were prepared for morphometric assessment and immunostaining for osteopontin (OPN) and bone sialoprotein (BSP). Transplanted cells were easily distinguished by fluorescent beads or by beta-galactosidase-positive expression and were distributed throughout the regenerating periodontal ligament (PL) and alveolar bone. At 1 week after wounding, animals treated with beta-galactosidase-positive cells exhibited a slightly higher percentage of labeled cells in the PL compared with the fluorescent bead-labeled cell implant group (2% vs. 1% respectively; P > 0.2). At Week 2 percentages of labeled cells were slightly increased in the regenerating PL (approximately 3% for both groups, P > 0.2). In regenerating alveolar bone at 1 week, animals that were treated with beta-galactosidase-positive cells and fluorescent bead-loaded cells exhibited approximately 30% and 25% of labeled cells respectively. At 2 weeks after wounding there was an increase in the percentage of transplanted beta-galactosidase-positive cells (approximately 39% at week 2; P < 0.05), but not of transplanted cells with fluorescent beads (approximately 25% at week 2). In sites with transplanted cells there were higher percentages of OPN positive and BSP positive cells in nascent bone and more newly formed bone than in controls (>40%; P < 0.05). Transplantation of beta-galactosidase-positive cells or cells loaded with fluorescent beads is a useful method for assessing the fate and differentiation of periodontal cells in vivo. Fluorescent beads, however, are diluted at mitosis and this method underestimates the percentage of transplanted cells. As transplanted periodontal cells in both groups promoted regeneration of alveolar bone, cell transplantation could improve the restoration of periodontium destroyed by periodontitis.
Anat Rec 2001 02 01
PMID:Transplantation of labeled periodontal ligament cells promotes regeneration of alveolar bone. 1116 14

Chronic degenerative disease of the mare's endometrium is characterised by changes in the uterine glands, including cystic dilation, hyperplasia and periglandular fibrosis. Endometrial biopsies were taken from 23 mares with different grades of endometrial degeneration. Solid structures were identified within the lumina of the uterine glands and shown to be calcified by histochemical staining. Most of them were not homogenous but composed of a mixture of mineral and organic substances. Further examinations of these mineralised structures by immunohistochemical methods revealed the presence of the non-collagenous matrix proteins osteopontin, osteonectin and bone sialoprotein, which are known to be involved in calcification processes such as urolithiosis. Osteopontin and bone sialoprotein were identified within the calculi, frequently arranged in concentric layers. Osteonectin was the only matrix protein that was also present in the glandular epithelium. Osteocalcin was not found in either the calculi or the glandular epithelial cells.
Vet Rec 2003 Dec 06
PMID:Mineralised deposits in the uterine glands of mares with chronic endometrial degeneration. 1469 74

Our previous studies suggested that a part of bone extracellular matrix (ECM) molecules are degraded and remodeled during embryonic bone formation. In contrast, little is known about ECM remodeling in postnatal appositional bone formation. The present study was designed to investigate expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during experimentally initiated appositional bone formation in rats. Expressions of ECM molecules, MMPs, and TIMPs were examined using in situ hybridization. Osteoblasts and osteocytes expressed MMP2 and -8, TIMP1, -2, and -3, as well as type I collagen, osteopontin, and osteocalcin in the course of the appositional bone formation, while they showed few transcripts of MMP13. The results indicated that while osteoblasts and osteocytes in the apposed bone produce ECM molecules, they degrade ECM molecules with MMPs and regulate the degradation by inhibiting the activity of MMPs using TIMPs. Osteoblasts and osteocytes may reorganize the ECM composition to mature the bone matrix in appositional bone formation.
Anat Rec A Discov Mol Cell Evol Biol 2004 Apr
PMID:Osteoblasts and osteocytes express MMP2 and -8 and TIMP1, -2, and -3 along with extracellular matrix molecules during appositional bone formation. 1505 53

The paranasal sinuses remain elusive both in terms of function and in the proximate mechanism of their development. The present study sought to describe the maxillary sinuses (MSs) in three species of callitrichid primates at birth, a time when secondary pneumatization occurs rapidly in humans. The MSs were examined in serially sectioned and stained slides from the heads of two Callithrix jacchus, one Leontopithecus rosalia, and two Saguinus geoffroyi. Specimens were examined microscopically regarding the distribution of osteoclasts and osteoblasts along the osseous boundaries of the MS and other parts of the maxillary bone. Selected sections were immunohistochemically evaluated for the distribution of osteopontin (OPN), which facilitates osteoclast binding. Taken together, OPN immunoreactivity and bone cell distribution suggested trends of bone resorption/deposition that were consistent among species for the superior (roof) and inferior (floor) boundaries of the MS. Expansion at the roof and floor of the MS appeared to correspond to overall vertical midfacial growth in callitrichids. Much more variability was noted for the lateral (alveolar) and medial (nasal walls) of the MS. Unlike the other species, the nasal wall of Saguinus was static and mostly composed of inferior portions of the nasal capsule that were undergoing endochondral ossification. The variation seen in the alveolar walls may relate to the presence or absence of adjacent structures, although it was noted that adjacency of deciduous molars influenced medial drift of the alveolar wall in Saguinus but not Leontopithecus. The results of this study are largely consistent with the "structural" or "architectural" hypothesis of sinus formation with respect to vertical MS enlargement, and the variable cellular/OPN distribution found along the nasal and alveolar walls was evocative of Witmer's (J Vert Paleontol 1997;17:1-73) epithelial hypothesis in revealing that most expansion occurred in regions unopposed by adjacent structures.
Anat Rec A Discov Mol Cell Evol Biol 2005 Aug
PMID:Secondary pneumatization of the maxillary sinus in callitrichid primates: insights from immunohistochemistry and bone cell distribution. 1598 87

Klotho-deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho-deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho-deficient mice-comparing the findings with those from their wild-type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho-deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho-deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho-deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization.
Anat Rec (Hoboken) 2008 Feb
PMID:Involvement of the klotho protein in dentin formation and mineralization. 1808 32

Development of the nasal capsule cartilages was studied in seven Geoffroy's tamarins (Saguinus geoffroyi), including one fetus, five neonates and one infant. Four additional postnatal specimens of the genus were studied (one 5-month-old and three adults) to determine the magnitude of postnatal expansion of the paranasal sinuses. Alcian blue histochemistry and osteopontin immunohistochemistry were employed in selected subadult specimens to characterize cartilage matrix. The fetal S. geoffroyi possesses a continuous nasal capsule, including a zona anularis; the primordial maxillary sinuses are surrounded by cartilage. Secondary pneumatization is in progress in all older specimens, which have sinuses that are more than twofold larger compared to that of the fetus. Results indicate that extensive ossification of the middle part of the nasal capsule (pars intermedia) is occurring in the perinatal timeframe, forming portions of the ethmoid bone. Anteriorly, the nasal capsule comprises isolated fragments in perinatal specimens, which are fewer and smaller in the infant and in a 5-month-old S. midas, and nearby multinucleate cells suggest that osteoclasts break apart these initially continuous elements. Fragments of the pars intermedia and the tectum nasi are found transiently between mucosa and the sites of secondary pneumatization. The maxillary sinus mucosa is highly vascular in most perinatal specimens. Histochemical and immunohistochemical findings show that cartilage of endochondral bones and non-ossifying parts are distinct in the perinatal time period. These results indicate that breakdown of the capsular cartilage precedes secondary pneumatization as previously suggested. There are portions of the cartilage of the recessus maxillaris and tectum nasi that transiently block mucosa from interfacing directly with bone. Vascularization may play a role in the breakdown of cartilages as well as the onset of secondary pneumatization. Since cartilage has the capacity to produce substances that trigger angiogenesis and bone resorption, further detailed characterization of the cartilage bordering sites of secondary pneumatization is merited.
Anat Rec (Hoboken) 2008 Nov
PMID:Fate of the nasal capsular cartilages in prenatal and perinatal tamarins (Saguinus geoffroyi) and extent of secondary pneumatization of maxillary and frontal sinuses. 1895 79


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