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PubMed2002

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

3T3 Cells↗
PubMed2002

Selective synthesis of bone morphogenetic proteins-1, -3, -4 and bone sialoprotein may be important for osteoinduction by Saos-2 cells.

An ability to induce new bone formation at a required site would represent a considerable advance in bone repair and tissue engineering. It has been shown that the healing of critical-size bone defects in rats can be augmented by extracts of Saos-2 cells. These human osteosarcoma cells uniquely contain a bone-inducing activity, whereas other human osteosarcoma cells, e.g., U-2 OS cells, cannot replicate the osteoinductive capacity. To understand the necessary components of the Saos-2 bone-inducing activity, this study compared osteoinductive Saos-2 cells with non-osteoinductive U-2 OS cells with respect to the synthesis of bone morphogenetic proteins (BMPs)-1, -2, -3, -4, -5, -6, and -7 and the non-collagenous matrix proteins bone sialoprotein (BSP), osteonectin (ON), osteopontin (OPN), and osteocalcin (OC). The main differences were abundant synthesis of BMP-1/tolloid, BMP-3, -4, and BSP by Saos-2 cells, but absence or reduced synthesis in U-2 OS cells. BMP-2 and -7 were present in low amounts in both cell types, while BMP-5 and -6 were more abundant in U-2 OS cells, suggesting that these BMPs were of lesser importance for the osteoinductivity of Saos-2 cells. However, a relatively high expression of BMP-3 and -4, together with BMP-1/tolloid, may be important for the osteoinductive capacity of Saos-2 cells. The inability of U2-OS cells to induce bone, despite expressing most of the BMPs, may be due to an insufficiency of tolloid, BMP-3 or -4, BSP, and/or other unknown factors. A better understanding of the necessary components of the Saos-2 cell bone-inducing agent may, in future, lead to clinically useful Saos-2 cell products for bone repair and tissue engineering.

Animals↗
PubMed2005

Detection of circulating stromal stem cells with osteogenic potential in the blood of coronary patients by laser flow cytometry.

The number of cells expressing antigen to osteonectin was appreciably increased in the blood of coronary patients, but no cells of this kind were detected in donors. The number of CD34+ stem cells in these patients virtually did not differ from that in normal subjects. A close relationship between atherosclerosis development and presence of stromal stem cells with osteogenic potential in the blood is hypothesized.

Antigens, CD34↗
PubMed1992

Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.

Pulp fibroblasts were isolated from human deciduous and supernumerary teeth and cultured in vitro. With continued culture in normal tissue-culture medium, six pulp fibroblast strains formed cell nodules after 10-15 days. By electron microscopy the nodules had matrix vesicles, and needle-shaped crystals associated with a dense network of collagen fibrils. The crystalline material exhibited a pattern consistent with hydroxyapatite when nodules were examined by X-ray diffractometry. Furthermore, the cells showed high levels of alkaline phosphatase activity, which could be increased more than seven-fold by the addition of 1,25(OH)2D3 (5 x 10(-9)-5 x 10(-8) M). In addition to the production of type I collagen, these cells also synthesized fibronectin and osteonectin. The formation of mineralized tissue nodules by pulp cells in vitro provides a useful system for study of the pathological calcification of pulp tissues.

Alkaline Phosphatase↗
PubMed1993

Influence of an intermittent compressive force on matrix protein expression by ROS 17/2.8 cells, with selective stimulation of osteopontin.

The purpose of this study was to determine the response of bone cells to physical stress. Intermittent compressive force (ICF) was applied to 13 kPa to subconfluent ROS 17/2.8 cells at 18 cycles/min. After 48 h of this application, the cells were labelled with [35S]-methionine or [32PO4]. Application of ICF over this time did not alter the synthesis of type I collagen, fibronectin or bone SPARC (osteonectin) compared to that of control cells. However, the activity of alkaline phosphatase was increased 1.5-fold, and the synthesis of a 32PO4-labelled, 75-kDa phosphoprotein, recognized as osteopontin by immunoprecipitation with specific antibodies, was increased 1.4-fold. Also, an increase in osteopontin mRNA starting within 12h of ICF application was observed. The selective increase in osteopontin expression associated with ICF may be important in the remodelling of bone tissues during growth and development and in response to functional forces.

Alkaline Phosphatase↗
PubMed1990

Purification of a calcium binding protein (rat SPARC) from primary Sertoli cell-enriched culture medium.

A protein with an apparent Mr of 43,000 was purified from Sertoli cell-enriched culture medium by sequential anion-exchange, gel permeation, C4 reversed-phase, and diphenyl reversed-phase HPLC. N-Terminal sequence analysis of this protein revealed a sequence of NH2-XPQTEAAEEMVAEETVV for the first 17 amino acids. Comparison of this sequence with existing protein data base maintained at the Protein Identification Resource revealed that it shares extensive identity with a previously described protein secreted by mouse embryo parietal endoderm, SPARC, which is equivalent to a protein secreted by a basement-membrane-producing tumor, BM-40; and a bone protein, osteonectin. This protein also possesses similar in vitro biological activity of SPARC in which it binds Ca2+. The possible physiological significance of this protein was discussed.

Amino Acid Sequence↗
PubMed1992

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.

Animals↗
PubMed1994

Bone-related matrix proteins expression in vitro and in vivo by marrow stromal cell line.

MBA-1, a bone marrow stroma-derived cell line, was transplanted in an ectopic site and formed endochondral bone. The ossicle developed through stages of cell proliferation, differentiated into a zone of hypertrophy and formed a chondroid-like area which further developed to primary mineralized bone. We explored the expression of various matrix proteins by MBA-1 cells in vitro and in the ossicle formed in vivo. MBA-1 cells constitutively expressed mRNAs encoding for collagen I, non-collagenous proteins and alkaline phosphatase. RNA extracted from the ossicle formed by these cells was expressed in a different pattern. The in vivo maturation of MBA-1 cells was accompanied by low expression of mRNA for procollagen alpha 2(I) and a marked increase in osteonectin and osteopontin mRNA levels. Thus, the ability to follow expression of these genes through bone formation in vivo has been demonstrated.

Actins↗
PubMed1994

Osteonectin/SPARC is a product of articular chondrocytes/cartilage and is regulated by cytokines and growth factors.

Rabbit articular chondrocytes maintained in monolayer, synthesized and secreted a 46 kDa protein into the culture medium. N-terminal sequence analysis and immunoprecipitation of the radiolabeled material revealed this protein to be osteonectin (ON)/SPARC, a protein previously shown to be present in bone. When chondrocytes were exposed to interleukin-1, a cytokine with matrix degradative properties, ON synthesis and secretion was greatly inhibited. However, this was specific to IL-1 since two other pro-inflammatory cytokines (tumor-necrosis factor-alpha and interleukin-6) with properties similar to IL-1, failed to cause any discernible effect on ON synthesis. Several growth factors (TGF-beta, PDGF, and IGF-1), that have been shown to stimulate other cartilage matrix macromolecular synthesis, also stimulated ON synthesis and were also able to reverse the inhibitory effect of IL-1 on ON synthesis. These observations were also demonstrated in explant cultures of cartilage. Our studies suggest that ON is a biosynthetic product of articular cartilage and could play a role in cartilage structure and/or function.

Amino Acid Sequence↗
PubMed1991

Down-regulation of SPARC/osteonectin/BM-40 expression in methylcholanthrene-induced fibrosarcomas and in Kirsten-MSV transformed fibroblasts.

SPARC (secreted protein acid and rich in cysteine), also known as osteonectin or BM-40, is a glycoprotein associated with the extracellular matrix of bone as well as with many soft tissues that produce extracellular matrix, including matrix-producing tumours. Northern and slot-blot analyses were used to study SPARC expression in tumours induced in vivo by methylcholanthrene (MCA) and in transformed cells induced in vitro by Kirsten-MSV and SV-40 infection. MCA-induced tumours expressed SPARC mRNA at quantitatively different levels. Fibroblasts transformed in vitro by Kirsten-MSV, and, to a lesser extent, by SV-40, showed reduced levels of SPARC mRNA expression compared with normal fibroblasts. Run-on assay indicated that transcription of SPARC was lower in the Kirsten-MSV transformed cells than in the normal parental fibroblast culture. However, SPARC mRNA in the transformed culture was as stable as that in normal culture. The difference, therefore, between levels of SPARC mRNA in transformed and normal culture was mainly due to different rates of transcription. Cloned cell lines derived from the Kirsten-MSV transformed culture also showed heterogeneous expression of SPARC: two lines had high and two had low expression of the gene. The level of mRNA correlated with that of the protein secreted. The SPARC expression might contribute to the malignant phenotype.

Animals↗
PubMed1991

Preferential adsorption of dentin and bone acidic proteins on the (100) face of hydroxyapatite crystals.

Interaction of bone and dentin proteins with minerals is an elementary step in the regulation of mineralization in these tissues. Adsorption of acidic non-collagenous proteins on hydroxyapatite was examined using fluorescence-labeled protein and synthetic hydroxyapatite. Phosphophoryn, bone Gla protein, osteonectin and bone small proteoglycan II were prepared and labeled with fluorescein. All of these proteins were adsorbed on hydroxyapatite with a dissociation constant on the order of 10(-7) M. The more acidic proteins had lesser binding capacities. Hydroxyapatite single crystals were incubated with labeled proteins and observed with a fluorescence microscope. Phosphophoryn and other acidic proteins were adsorbed preferentially on the (100) face of the crystal. This preferential adsorption of the acidic proteins may be responsible for the morphogenesis of biological hydroxyapatite.

Animals↗
PubMed1988

Molecular analysis of the cDNA for human SPARC/osteonectin/BM-40: sequence, expression, and localization of the gene to chromosome 5q31-q33.

Human cDNA clones encoding the extracellular calcium-binding, acidic glycoprotein known as SPARC, osteonectin, or BM-40 were isolated from a placental cDNA library. Two polyadenylated transcripts of 2.2 and 3.0 kb were detected in human tissues and cultured cells by Northern blot analysis, and cDNAs for both transcripts were characterized. The 2133-bp sequence of the more abundant (major) transcript contains an open reading frame for 303 amino acids. The deduced polypeptide has extensive amino acid sequence identity with mouse SPARC. The larger and minor 3.0-kb cDNA has an identical coding region but utilizes a downstream polyadenylation signal. Gene localization studies have revealed a single chromosomal site at 5q31-q33 by somatic cell hybrid analysis and in situ chromosomal hybridization. Furthermore, pulsed-field gel electrophoresis of human genomic DNA cleaved with different rare-cutting restriction enzymes and hybridized with SPARC cDNA probes revealed single or double fragments of less than 50 to about 150 kb. The evidence is consistent with a single locus for SPARC in humans. The gene was found to be differentially expressed in many human tissues and in an osteogenic sarcoma, but not in other transformed cells.

Amino Acid Sequence↗
PubMed1991

A linkage map spanning the locus for diastrophic dysplasia (DTD).

Diastrophic dysplasia (DTD) is an autosomal recessive osteochondrodysplasia. Patients have short-limbed short stature and suffer from generalized joint dysplasia. We have recently mapped DTD to the distal long arm of chromosome 5. Here we report the localization of DTD in relation to 16 polymorphic markers on distal 5q. No recombinations occurred with two loci, D5S72 and D5S66. One presumptive candidate gene, osteonectin (SPARC), could be excluded on the basis of three recombinations with the DTD locus. Multipoint linkage analysis performed against a fixed order of markers placed DTD between glucocorticoid receptor (GRL) and SPARC favored by the odds of 33:1 over the next best location of DTD between D5S72 and D5S55. The sex-averaged distance between the definite flanking markers, GRL and D5S55, is 17.5 cM. From previously reported data on the physical localization of markers, we conclude that the DTD locus is in 5q31-q34.

Alleles↗
PubMed1996

Renaturation of SPARC expressed in Escherichia coli requires isomerization of disulfide bonds for recovery of biological activity.

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin and BM-40) belongs to a group of secreted macromolecules that modulate cellular interactions with the extracellular matrix. During vertebrate embryogenesis, as well as in tissues undergoing remodeling and repair, the expression pattern of SPARC is consistent with a fundamental role for this protein in tissue morphogenesis and cellular differentiation. Human SPARC was cloned by the polymerase chain reaction from an endothelial cell cDNA library and was expressed in Escherichia coli as a biologically active protein. Two forms of recombinant SPARC (rSPARC) were recovered from BL21(DE3) cells after transformation with the plasmid pSPARCwt: a soluble, monomeric form that is biologically active (Bassuk et al., 1996, Archiv. Biochem. Biophys. 325, 8-19), and an insoluble form sequestered in inclusion bodies. Aggregated rSPARC was unfolded by urea treatment, purified by nickel-chelate affinity chromatography, and renatured by gradual removal of the denaturant. Proper isomerization of the disulfide bonds was achieved in the presence of a glutathione redox couple. After final purification by high resolution gel filtration chromatography, a monomeric form of rSPARC displaying biological activity was obtained. The recombinant protein inhibited the spreading and synthesis of DNA by endothelial cells, two properties characteristic of the native protein. We conclude that the information for the correct folding of rSPARC resides in the primary structure of the protein, and suggest that post-translational modifications are required neither for folding nor for biological activity.

Animals↗
PubMed1985

Matrix protein profiles in calf bone development.

The major noncollagenous proteins (NCP) of the mineralized extracellular matrix of bovine bone were present from early fetal life through adulthood. The alpha 2-HS-glycoprotein was the dominant bone NCP at 2 months in utero and then diminished with advancing fetal age. Osteocalcin levels were quite low in early fetal bone, became elevated near term, reached a maximum shortly after birth, and dropped again in adult bone. Osteonectin levels were relatively constant at all stages of bone development, but somewhat lower values were seen in woven bone than in lamellar bone matrix. For a given fetus, NCP profiles were identical in both flat and long bones. The 24,000 dalton phosphoprotein was significantly elevated in fetal secondary spongiosa, bony areas known to be undergoing extensive resorption. Adult bone matrix contained greater quantities of lower and intermediate molecular weight NCPs compared to actively forming fetal bone.

Animals↗
PubMed2006

Characterization of osteoblasts derived from bone marrow stromal cells in a modified cell culture system.

Bone marrow is a complex tissue composed of hematopoietic and stromal stem cells with the potential to differentiate into adipogenic, fibroblastic, reticular, osteogenic and chondrogenic lineages. Identification of differentiation markers during transformation of stromal cells into osteoblasts in a time-dependent manner may be informative for cell-based tissue engineering. Therefore, we investigated the effects of osteogenic medium (OM) on the proliferation and differentiation of rat bone marrow stromal cells (BMSCs). BMSCs from adult male rat tibia and femur were collected and cultured in alpha-MEM medium with 10% fetal bovine serum, penicillin, streptomycin and gentamycin. After three days of culture, the medium covering the adherent cells in culture was changed to OM containing dexamethasone, Na-beta-glycerophosphate and ascorbic acid. As a control, cell culture was also continued in the original medium for the same time period. Differentiated osteoblast cells were collected after 7, 10, 14, 21 and 30 days of culture, fixed with 4% paraformaldehyde and their immunolabelling for osteoblast markers osteonectin (ON) and osteocalcin (OC) was assessed using an indirect immunoperoxidase technique. Immunolabelling of ON and OC was detectable from day 10 of culture, began to increase on day 14, and increased steadily through to day 21. Labelling was highest on day 30 and was more intense in cells cultured with OM compared to the culture without OM. The control cells cultured in the absence of OM produced negligible levels of both markers. In conclusion, our culture system facilitated differentiation of BMSCs into osteoblasts featuring osteoblast markers, and these cells may be useful in autologous bone implant for the treatment of bone wound healing.

Animals↗
PubMed2005

Amelogenin: lessons from evolution.

Amelogenin plays a crucial role in enamel structure and mineralization, but the function of its various domains is far to be understood. Evolutionary analysis seems to be a promising way to approach structure/function relationships. In this paper, we review the knowledge of amelogenin with a particular focus on what we have learnt from evolution, and we bring new data on the origin and evolution of this molecule. The comparison of amniote (reptiles and mammals) amelogenin sequences reveals that, in contrast to the well-conserved C- and N-terminal domains, the central region (most of exon 6) is highly variable. The evolutionary analysis indicates that it was created by repeated insertion of three amino acids (triplets ProXGlu or ProXX). In several mammalian lineages a new run of triplet insertions and deletions has occurred independently in a locus considered a hot spot of mutation for mammalian amelogenin. In lizard and snake amelogenin evolves rapidly. Sequence alignment reveals that several residues in the N- and C-terminal regions were kept unchanged during 250 million years (MY), proving their importance for amelogenin structure and function. This alignment permits a rapid validation of the amelogenin mutations in human. Genome sequencing and gene mapping permitted to refine the amelogenin story, in relation to the common location (chromosome 4 in human) of several genes coding for dental proteins and SPARCL1, a SPARC (osteonectin) relative. Amelogenin shares a similar organisation with these genes and a blast search in databanks indicates a strong relationship between amelogenin, ameloblastin and enamelin. Taken together these data suggest that amelogenin could have originated from either ameloblastin or enamelin, themselves being created from SPARCL1, which itself originated from a SPARC duplication, 600 millions years ago.

Amelogenesis↗
PubMed2005

RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation.

OBJECTIVE: The structural organization of oral soft tissue and its relationship with highly calcified teeth are difficult to preserve unless tissues are decalcified, paraffin embedded and subsequently sectioned. However, enamel decalcification time and its negative impact on RNA integrity makes it difficult to effectively analyse in situ gene expression. This study examined the impact of microwave-enhanced decalcification on processing time, RNA integrity and detection of in situ mRNA expression in hard and soft tissue for cell type specific markers of Keratinocyte growth factor receptor, Scleraxis and Osteonectin. DESIGN: Maxillas and mandibles were obtained from three male Wistar strain rats. Right side tissues were decalcified using a microwave plus 10% EDTA solution (M+) while left side tissues were decalcified in 10% EDTA solution alone (M-). RESULTS: Microwave use reduced decalcification time by up to 50% and had no significant impact on morphology, RNA quality and in situ detection of gene expression relative to the M-group. CONCLUSIONS: In situ RT-PCR gene expression of microwave decalcified paraffin-embedded oral tissues is an effective technique to localize in situ gene expression while maintaining excellent soft and hard tissue architecture.

Animals↗