Immunolocalization Of Osteopontin Research Articles

Low concentrations of grape seed extract maintain osteoblast morphology, cell adhesion, and mineralization.

The increase in life expectancy has led to a higher incidence of osteoporosis, characterized by an imbalance in bone remodeling. Several drugs are used for its treatment, but most promote undesirable side effects. The present investigation evaluated the effects of two low concentrations of grape seed extract (GSE) rich in proanthocyanidins on MC3T3-E1 osteoblastic cells. The cells were cultured in an osteogenic medium and divided into control (C), 0.1 µg/mL GSE (GSE0.1), and 1.0 µg/mL GSE (GSE1.0) groups to evaluate cell morphology, adhesion, and proliferation, in situ alkaline phosphatase (ALP) detection, mineralization and immunolocalization of osteopontin (OPN). The data obtained were analyzed by statistical tests for a significance of 5%. Cell morphology was maintained with both GSE concentrations, whereas cell adhesion significantly increased within three days in all groups. Cell proliferation increased significantly at seven days of culture, followed by a significant decrease in all experimental periods, with no statistical difference among them. In situ detection of ALP and mineralization increased with time, but within each period, no statistical differences among groups were observed. The expression of osteopontin was distributed regularly with more intensity after 24 hours in the GSE0.1 group. After three days, OPN expression was more intense in the control group, followed by GSE0.1 and GSE1.0 groups. Data obtained suggest that low concentrations of GSE do not affect the morphology and may stimulate the functional activity of osteoblastic cells.

Immunolocalization of osteopontin in dysplasias and squamous cell carcinomas arising from oral epithelium.

Background:Early detection of oral squamous cell carcinoma (OSCC) remains one of the most efficient ways to ensure patient survival and improved quality of life. Although specific biomarkers related to OSCC have been investigated, a useful biomarker that assesses the transition potential of potentially malignant lesion to OSCC remains to be found. Osteopontin (OPN) has been recognized as an important factor in tumorigenesis and their expression in OSCC have been investigated earlier. In the present study, evaluation of OPN expression in premalignant and malignant lesions has been carried out to assess their possible role as a biomarker in the early diagnosis and prognosis of OSCC.Objectives:The objective of this study is to evaluate the role of OPN as a biomarker in the diagnosis and prognosis of OSCC.Materials and Methods:The study group consisted of archival paraffin-embedded blocks of ten cases each of varying grades of OSCC, oral epithelial dysplasias and epithelial hyperplasias. Sections were subjected to immunohistochemical staining for the biomarker OPN.Results:A positive OPN expression was noticed in epithelial dysplasias and SCC arising from the oral epithelium. A progressive increase in the intensity of staining was seen with increasing grades of dysplasias and a decrease in OPN expression with an increase in grades was observed in OSCC.Conclusion:The expression of OPN in full thickness of epithelium in severe dysplasias, carcinoma in situ, and in the superficial epithelium of OSCC suggest the possibility of considering OPN expression in full epithelial thickness in dysplasias as an indicator for malignant transformation.

The effect of a nanothickness coating on rough titanium substrate in the osteogenic properties of human bone cells

This study evaluated the effect of a bioactive ceramic coating, in the nanothickness range, onto a moderately rough surface on the osteogenic behavior of human bone cells. The cells were harvested from the mandibular mental region and were cultured over Ti-6Al-4V disks of different surfaces: as-machined (M), alumina-blasted/acid etched (AB/AE), and alumina-blasted/acid-etched + 300-500 nm thickness amorphous Ca- and P-based coating obtained by ion beam-assisted deposition (Nano). The culture was then evaluated regarding cell viability, adhesion, morphology, immunolocalization of osteopontin (OPN) and alkaline phosphatase (ALP). The results showed that the surface treatment did not interfere with cell viability. At 1 day, AB/AE and Nano showed higher adhesion than the M surface (p < 0.001). Higher adhesion was observed for the M than the Nano surface at 7 days (p < 0.005). The percentage of cells showing intracellular labeling for OPN at day 1 was significantly higher for the Nano compared to M surface (p < 0.03). The percentage of ALP intracellular labeling at 7 days was significantly higher for the AB/AE compared to the M surface (p < 0.0065); no differences were detected at 14 days. Our results suggest that the presence of a thin bioactive ceramic coating on a rough substrate did not favor the events related to in vitro osteogenesis. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Immuno-Localization of CD44 and Osteopontin in Developing Human Kidney

CD44 is observed in ureteric bud structures and is implicated in branching morphogenesis during early mouse renal development. Healthy adult kidney demonstrates minimal CD44, but CD44 is up-regulated in renal diseases. CD44 may mediate binding of calcium oxalate crystals to tubular epithelia via the ligands osteopontin (OPN) and hyaluronan. Because 15% of premature infants develop nephrocalcinosis, developmental tubular CD44 expression might promote nephrocalcinosis. We studied CD44 and OPN immuno-localization in developing human kidney by immunohistochemical analysis. Human renal tissue between 18 and 40 wk of gestation showed CD44 immuno-localization in ureteric buds, with staining decreasing with increasing gestational age; CD44 was rarely observed in developing renal tubules. OPN was diffusely observed in proximal tubules, rarely observed in distal tubules, ureteric buds or metanephric structures. These data support the role of CD44 in early human nephron formation and branching morphogenesis. Rare CD44 staining in developing tubular epithelium suggests no role for CD44 in promoting calcium oxalate adherence to tubular epithelia in premature infants. Immuno-localization of OPN in tubules supports its role in tubular differentiation, but OPN does not seem to be necessary during early nephron formation.

Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures

As part of ongoing studies aimed at clarifying the early events of bone matrix deposition and mineralization, we have characterized primary osteoblast cultures using ultrastructural and immunocytochemical methods. Osteogenic cells were isolated by sequential enzymatic digestion of newborn rat (2–4-day-old) calvariae and grown for periods of 7 to 28 days on polystyrene, Thermanox plastic, or sputtered titanium. Bone-like nodules, showing a stratified organization of cells and collagen, were examined by scanning and transmission electron microscopy, and further characterized for mineral by backscattered electron imaging and X-ray microanalysis. Colloidal gold immunocytochemistry was used to examine the distribution of osteopontin in these nodules. Cells at the surface of the nodules were rounded, while those within the nodules generally appeared more flattened. Both cell types, particularly at early culture intervals, exhibited well-developed protein synthetic organelles. Collagen fibrils were present between the cell layers and some individual fibrils appeared mineralized. Aggregates of needle-shaped crystallites were sometimes apposed to the cell surface, frequently within invaginated regions of the cell membrane, while other mineralized masses of various sizes were present within the collagenous scaffolding. The periphery of the mineralized masses was often delimited by an electron-dense, lamina limitans-like layer. Focal accumulations and/or a more complete layer of afibrillar, mineralized organic matrix were sometimes observed at the interface between the cells and the surface of the culture dish. Osteopontin was immunodetected over the afibrillar and collagenous mineralized matrix throughout the cultures and, in some cases, labeling was concentrated over the peripheral, electron-dense material delimiting the mineralized masses. In conclusion, these data indicate that calvaria-derived osteoblasts produce an extracellular matrix with structural and compositional similarities to bone. Although not a regular observation, the accumulation of osteopontin on the surface of the culture substrate and at the periphery of masses of mineralized matrix may be analogous to what takes place in vivo at naturally occurring bone interfaces. © 1996 Wiley-Liss, Inc.

Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures.

As part of ongoing studies aimed at clarifying the early events of bone matrix deposition and mineralization, we have characterized primary osteoblast cultures using ultrastructural and immunocytochemical methods. Osteogenic cells were isolated by sequential enzymatic digestion of newborn rat (2-4-day-old) calvariae and grown for periods of 7 to 28 days on polystyrene, Thermanox plastic, or sputtered titanium. Bone-like nodules, showing a stratified organization of cells and collagen, were examined by scanning and transmission electron microscopy, and further characterized for mineral by backscattered electron imaging and X-ray microanalysis. Colloidal gold immunocytochemistry was used to examine the distribution of osteopontin in these nodules. Cells at the surface of the nodules were rounded, while those within the nodules generally appeared more flattened. Both cell types, particularly at early culture intervals, exhibited well-developed protein synthetic organelles. Collagen fibrils were present between the cell layers and some individual fibrils appeared mineralized. Aggregates of needle-shaped crystallites were sometimes apposed to the cell surface, frequently within invaginated regions of the cell membrane, while other mineralized masses of various sizes were present within the collagenous scaffolding. The periphery of the mineralized masses was often delimited by an electron-dense, lamina limitans-like layer. Focal accumulations and/or a more complete layer of afibrillar, mineralized organic matrix were sometimes observed at the interface between the cells and the surface of the culture dish. Osteopontin was immunodetected over the afibrillar and collagenous mineralized matrix throughout the cultures and, in some cases, labeling was concentrated over the peripheral, electron-dense material delimiting the mineralized masses. In conclusion, these data indicate that calvaria-derived osteoblasts produce an extracellular matrix with structural and compositional similarities to bone. Although not a regular observation, the accumulation of osteopontin on the surface of the culture substrate and at the periphery of masses of mineralized matrix may be analogous to what takes place in vivo at naturally occurring bone interfaces.

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, α2HS-glycoprotein and alkaline phosphatase

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and α 2HS-glycoprotein ( α 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. α 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and α 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that α 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.

Immunolocalization of osteopontin, osteocalcin, and dentin sialoprotein during dental root formation and early cementogenesis in the rat

Using immunohistochemical methods we studied the tissue localization of the extracellular matrix proteins osteopontin (OPN), osteocalcin (OC), and dentin sialoprotein (DSP) during the formation of acellular and cellular cementum in newly born rats. In the layer of acellular cementum of developing incisor and molar teeth we found a very strong staining for OPN but not for DSP or OC. Many cells immediately adjacent to acellular cementum and PDL cells were also positive for OPN but not for DSP or for OC. In contrast, cellular cementum in molar teeth stained strongly for OPN and OC but not for DSP. Consistent with these observations, the cells engaged in the formation of cellular cementum (cementoblasts and cementocytes) reacted strongly for OPN and OC but not for DSP. In advanced stages of dentinogenesis, both crown and root odontoblasts and dentin stained for OPN, OC, and DSP. Cells and matrices of surrounding alveolar bone stained for OPN and OC but not for DSP. We conclude that cementoblasts and cementocytes of cellular cementum produce OPN and OC but not DSP and thus express an osteoblast-like, not an odontoblast-like, phenotype. The cells responsible for the production of acellular cementum are likely cells of the PDL in close contact with the dental root surface. These fibroblast-like cells express OPN but not OC or DSP and accordingly express only a partial osteoblastic phenotype.

High-resolution immunolocalization of osteopontin and osteocalcin in bone and cartilage during endochondral ossification in the chicken tibia.

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.