Non-mineralized Tissues Research Articles

A Homeodomain Protein Binding Element in the Bone Sialoprotein Promoter is Critical for Tissue-Specific Expression in Bone

Bone sialoprotein (BSP) expression is restricted to cells associated with the mineralization of bones and teeth. We previously identified a homeodomain binding element in a 2.5 kb fragment of the murine Bsp promoter that is required for osteoblast-selective expression in cell culture. To examine the role of this element (called OSHE1; osteoblast-specific homeodomain element 1) in the tissue-specific expression of Bsp in vivo, we generated transgenic mice using the wild-type 2472 bp promoter or the same promoter containing a 2 bp mutation in OSHE1. Promoter constructs driving both luciferase and lacZ reporter genes were microinjected into fertilized eggs from (C57BL/6 X SJL)F1 mice. Four lines containing the wild-type promoter and 5 lines containing the mutated promoter were established, and the tissue specificity of &#103 -galactosidase staining and luciferase expression was examined. &#103 -gal staining was observed in osteoblasts of calvaria and trabecular regions of tibia and femur in 12-day-old mice while chondrocytes, kidney, heart, muscle, spleen, liver, skin, stomach, and lung were negative. Whole tissue luciferase activity was also much higher in mineralized tissues although some soft tissue expression was detected. In contrast, analysis of OSHE1 mutant lines revealed expression of luciferase and &#103 -gal in kidney, skin, liver, and lung. &#103 -gal expression in these tissues was restricted to specific cell populations. Trabecular regions were devoid of &#103 -gal staining in the tibia and femur of the mutant mice, while staining was seen in the chondrocytes. We therefore hypothesize that the OSHE1 site is involved in both the expression of Bsp in mineralizing tissues and the suppression of transcription in nonmineralizing tissues.

A Homeodomain Protein Binding Element in the Bone Sialoprotein Promoter is Critical for Tissue-Specific Expression in Bone

Bone sialoprotein (BSP) expression is restricted to cells associated with the mineralization of bones and teeth. We previously identified a homeodomain binding element in a 2.5 kb fragment of the murine Bsp promoter that is required for osteoblast-selective expression in cell culture. To examine the role of this element (called OSHE1; osteoblast-specific homeodomain element 1) in the tissue-specific expression of Bsp in vivo, we generated transgenic mice using the wild-type 2472 bp promoter or the same promoter containing a 2 bp mutation in OSHE1. Promoter constructs driving both luciferase and lacZ reporter genes were microinjected into fertilized eggs from (C57BL/6 X SJL)F1 mice. Four lines containing the wild-type promoter and 5 lines containing the mutated promoter were established, and the tissue specificity of g -galactosidase staining and luciferase expression was examined. g -gal staining was observed in osteoblasts of calvaria and trabecular regions of tibia and femur in 12-day-old mice while chondrocytes, kidney, heart, muscle, spleen, liver, skin, stomach, and lung were negative. Whole tissue luciferase activity was also much higher in mineralized tissues although some soft tissue expression was detected. In contrast, analysis of OSHE1 mutant lines revealed expression of luciferase and g -gal in kidney, skin, liver, and lung. g -gal expression in these tissues was restricted to specific cell populations. Trabecular regions were devoid of g -gal staining in the tibia and femur of the mutant mice, while staining was seen in the chondrocytes. We therefore hypothesize that the OSHE1 site is involved in both the expression of Bsp in mineralizing tissues and the suppression of transcription in nonmineralizing tissues.

Post-Cambrian closure of the deep-water slope-basin taphonomic window

Exceptional faunas (Konservat-Lagerstatten that preserve traces of volatile nonmineralized tissues) are statistically overabundant in the Cambrian Period; almost all examples preserved in continental-slope and shelf-basin environments are of this age. The hypothesis that an increase in the amount and complexity of bioturbation was an important agent in the elimination of this deep-water slope-basin taphonomic window is supported. Post-Cambrian ichnofaunal assemblages contain a higher proportion of pascichnia and agrichnia, ethologies produced by a mobile infauna. They also illustrate the lateral partitioning of organisms into different environmental niches; both opportunistic and equilibrium infaunas occur in low-oxygen environments in which the preservation of nonbiomineralized tissues was favored. Direct consumption of carcasses by bioturbating organisms was less important than changes to sediment properties as a result of bioturbation, notably enhanced microbial degradation of reactive organic matter, increased permeability, and the disruption of geochemical gradients necessary for mineral authigenesis.

Developmental and TGF-β-mediated regulation of Ank mRNA expression in cartilage and bone

ObjectivesAnk encodes a transmembrane protein that is involved in pyrophosphate (PPi) transport and mutations in the Ank gene have been associated with pathological mineralization in cartilage and bone. To understand how Ank works in normal skeletal development it is also important to know which cells within the developing skeleton express Ank. To this end, we examined the expression pattern of Ank mRNA during mouse embryonic development as well as in mouse hind limb joints with emphasis on the period when articular cartilage forms. Since it was previously shown that TGF-β regulates PPi transport in cells in culture, we also tested the hypothesis that TGF-β regulates Ank expression.Methods The localization of Ank mRNA was determined by radioactive in situ hybridization in E15.5 and E17.5 mouse embryos as well as in 1 and 3 week post-natal mice. Ank expression was compared to that of other cartilage markers. In situ hybridization and semi-quantitative RT-PCR were used to determine the effects of TGF-β on Ank expression in metatarsal organ cultures.Results Ank expression was detected at high levels at sites of both endochondral and intramembranous bone development. In endochondral bones, expression was detected in a subset of hypertrophic cells at ossification centers. Expression was also detected in osteogenic/chondrogenic cells of the perichondrium/periosteum lining the metaphysis, an area associated with the formation and extension of the bone collar. High levels of expression were also detected in non-mineralized tissues of the skeletal system including tendons and the superficial layer of the articular cartilage. Treatment with TGF-β resulted in an approximately four-fold induction of Ank mRNA in prehypertrophic chondrocytes and perichondrium of metatarsal cultures.Conclusions The expression pattern of Ank suggests an important role both in inhibiting and regulating mineralization in the developing skeletal system. In addition, TGF-β1 is able to mediate Ank mRNA expression in chondrocytes suggesting a possible role for TGF-β and Ank in the regulation of normal mineralization. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

The Human Tuftelin Gene and the Expression of Tuftelin in Mineralizing and Nonmineralizing Tissues

Tuftelin has been suggested to play an important role during the development and mineralization of enamel, but its precise function is still unclear. This article reviews major milestones in the discovery, structural characterization, expression, localization, and conservation of tuftelin in different vertebrate species. It focuses on the structure of the human tuftelin gene, which has recently been deciphered [12]. It describes the exon-intron organization, sizes and structure, the promoter structure, and the newly discovered alternatively spliced human tooth-bud tuftelin mRNA transcripts. It also examines information on the structural motifs in the human-derived tuftelin protein and how they relate to tuftelin from other species. It reviews our recent results on the transcription of tuftelin mRNA and protein expression in several nonmineralizing soft tissues, using reverse-transcription polymerase chain reaction (RT-PCR) followed by DNA cloning and sequencing, indirect immunohistochemistry, immunohistochemistry combined with confocal microscopy, and in situ hybridization. These results and earlier Northern blot results show that tuftelin, in addition to being expressed in the developing and mineralizing tooth, is also expressed in several nonmineralizing soft tissues, suggesting that tuftelin has a universal function and/or a multifunctional role.

The human tuftelin gene: cloning and characterization

Tuftelin has been suggested to play an important role during the development and mineralization of enamel. We isolated the full-length human tuftelin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (5′ RACE and 3′ RACE) methods. Sequence analysis of the tuftelin cDNA revealed an open reading frame of 1170 bp encoding a 390 amino acid protein with a molecular mass of 44.3 kDa and an isoelectric point of 5.7. The human tuftelin protein shares 89 and 88% amino acid sequence identity with the bovine and mouse tuftelin, respectively. It contains a coiled-coil region, recently reported to be involved with tuftelin self-assembly and with the interaction of tuftelin with TIP39 (a novel tuftelin interacting protein). Detailed DNA analysis of the cloned genomic DNA revealed that the human tuftelin gene contains 13 exons and is larger than 26 kb. Two alternatively spliced tuftelin mRNA transcripts have now been identified in the human tooth bud, one lacking exon 2, and the other lacking exon 2 and exon 3. Primer extension analysis, corroborated by RT-PCR and DNA sequencing, revealed multiple transcription initiation sites. The cloned 1.6 kb promoter region contained several GC boxes and several transcription factor binding sites such as those for activator protein 1 and stimulatory protein 1. Our blast search of the human and mouse expressed sequence tag data bases, as well as our RT-PCR and DNA sequencing results, and a previous study using Northern blot analysis revealed that tuftelin cDNA sequences are also expressed in normal and cancerous non-mineralizing soft tissues, suggesting that tuftelin has a universal function. We have now identified and characterized different alternatively spliced mouse tuftelin mRNAs in several non-mineralizing tissues. These results provide an important baseline for future understanding of the biological role of tuftelin.

The relative roles of intragenic polymorphisms of the vitamin d receptor gene in lumbar spine degeneration and bone density.

A retrospective cohort study. To compare the magnitudes of the associations of TaqI polymorphisms of the vitamin D receptor gene with bone density and lumbar spine degeneration in the same sample. Vitamin D receptor gene variations are associated with osteoporosis, osteoarthritis, and disc degeneration. Their role in these conditions remains poorly understood. Bone density of the spine and femur were determined through DEXA, and lumbar disc degeneration was determined from magnetic resonance imaging assessments of signal intensity, disc narrowing, bulging, anular tears, herniations, and osteophytes. Associations between these measures and TaqI polymorphisms of the coding region of the Vitamin D receptor locus were examined in a population-based sample of 142 men. The strongest associations were with signal intensity and anular tears, which were worse for the subjects with tt genotypes than for those with TT genotypes in the L4-S1 spine discs. Conversely, the prevalences of disc bulges and osteophytes were lowest for the tt genotype. Bone density, disc height, and herniations did not differ significantly by genotype. The strongest association of Vitamin D receptor TaqI polymorphisms with degeneration in nonmineralized connective tissues suggests that the underlying mechanism of TaqI polymorphisms is not specific to bone. This study demonstrated for the first time that those with the tt genotype had more anular tears than those with the TT genotype, a finding that should stimulate further analyses of this gene in conditions that result in back pain. The apparent discrepancies of the associations of the tt genotype with lower signal intensity and more anular tears, but less bulges and osteophytes, could be explained if bulging and osteophytes primarily represented remodeling related to lifetime physical loading.

Regeneration of Class III furcation defects with basic fibroblast growth factor (b-FGF) associated with GTR. A descriptive and histometric study in dogs.

The poor predictability of periodontal regenerative treatment of Class III furcation defects stimulates the study of alternatives to improve its results, such as the use of polypeptide growth factors. The objective of this study was to evaluate, both histologically and histometrically, the effects of topical application of basic fibroblast growth factor (b-FGF) associated with guided tissue regeneration (GTR) in the treatment of Class III defects surgically induced in dogs. All second and fourth premolars of 5 mongrel dogs were used and randomly assigned to one of three treatment groups: group 1 (control), treated with scaling and root planing, tetracycline hydrochloride (125 mg/ml) conditioning, and GTR with a collagen membrane; group 2, same treatment as group 1 plus 0.5 mg of b-FGF; group 3, same treatment as group 1 plus 1.0 mg of b-FGF. After a 90-day healing period, routine histologic processing and staining with hematoxylin and eosin and Masson trichrome were performed. The descriptive analysis indicated better regenerative results in both groups treated with b-FGF while the histometric data, analyzed by means of analysis of variance (ANOVA), showed greater filling of the defects in group 2 in comparison to the defects in groups 3 and 1, respectively, which was represented by a smaller area of plaque-occupied space (P = 0.004) as well as a greater amount of newly formed cementum (P = 0.002). These results indicate that b-FGF, especially in smaller doses, may enhance the regenerative results in Class III furcation lesions, leading to greater filling of these defects with both mineralized and non-mineralized tissues.

ROLE OF MR IMAGING IN SPORTS MEDICINE RESEARCH: Basic Science and Clinical Research Studies

The advent and advancement of MR imaging have provided an entire new dimension for medical imaging. MR imaging has been especially useful because of its capacity to image nonmineralized tissues with a very high degree of resolution. Although modalities such as ultrasound and scintigraphy have proven useful for specific purposes, it is MR imaging that has the most utility and capabilities, especially in the area of sports-induced injuries. The technology associated with MR imaging has expanded greatly, and it continues to evolve at a rapid pace. The result has been an ever-increasing diagnostic capability that has become more economic with time. As described previously, MR imaging is gaining importance in the area of comparative medicine for animal athletes as well. It is also interesting to note that MR imaging now has a greater potential for monitoring physiological and biochemical changes as well as anatomic ones. Some newer MR units actually include physiologic data acquisition components. Consequently, new bioassays and nondestructive tissue tests can be performed to further understand the molecular biology and ongoing cellular processes in any given condition. Coupled with MR spectroscopy, the enhanced MR techniques should continue to contribute to the overall information that will be integrated into the training and rehabilitation of patients with sports-induced inflammation and injuries. The authors support and encourage ongoing efforts in the area of MR imaging research, both basic science and clinical studies.

Expression and localization of membrane type 1 matrix metalloproteinase in tooth tissues.

Matrix metalloproteinases (MMPs) have been detected in forming dental enamel and are thought to play an important role during enamel biomineralization. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane bound member of the MMP gene family that has previously been shown to be expressed by cells associated with bone and cartilage formation (osteoclasts, osteoblasts and chondrocytes). Thus, we asked if MT1-MMP was also expressed by the cells responsible for the formation of enamel and dentin. A porcine MT1-MMP cDNA composed of 3284 bp was isolated from an enamel organ-specific cDNA library. Multiple tissue Northern blot analysis revealed that the MT1-MMP message was expressed highly in the enamel organ and pulp organ when compared to the expression levels observed in other non-mineralizing tissues. Northern blot analysis of stage-specific enamel organs (early secretory, late secretory, or maturation stage) and their corresponding pulp organs revealed that MT1-MMP expression increased as the dentin matured. In the enamel organs, however, the MT1-MMP message level became reduced only during the late secretory stage. Immunohistochemical analysis showed that MT1-MMP was present on the surface of the cells (ameloblasts and odontoblasts) responsible for dentin and enamel formation. Thus, MT1-MMP is highly expressed in developing tooth tissues and may play a role in the biomineralization of enamel and dentin.

Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes.

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca2+ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro-osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.

Increased Turnover of Small Proteoglycans Synthesized by Human Osteoblasts During Cultivation with Ascorbate and β-Glycerophosphate.

The small proteoglycan decorin had been localized previously at the d-band in the gap zone of collagen fibrils in nonmineralizing tissues. In bone matrix this zone is proteoglycan free and is at least in some species the place where mineralization along collagen fibrils starts. To study the metabolism of the small proteoglycans decorin and biglycan under mineralizing conditions, osteoblasts from human nasal bone were cultured for several weeks in the presence or absence of β-glycerophosphate and ascorbate. An immediate consequence of the treatment was a reduced expression of decorin, as judged by immune precipitation, whereas the biosynthesis of biglycan was not affected. Pulse-chase experiments were performed with osteoblasts embedded in floating type I collagen gels. In the presence of β-glycerophosphate and ascorbate, a more rapid turnover of both proteoglycans was noted; the one of biglycan reached statistical significance. Indirect evidence for an enhanced rate of proteoglycan endocytosis was obtained. This effect was not seen in cultured skin fibroblasts. Thus, osteoblasts respond rapidly to mineralizing conditions with alterations of small proteoglycan biosynthesis and turnover.

Collagen fibrillar structure in mineralized and nonmineralized tissues

The four most important recent developments have been the following determinations: first, that collagen molecules have considerable flexibility so that in regions devoid of glycine—proline—proline or glycine—proline—hydroxyproline in each chain, substantial helix distortion can be allowed; second, that this flexibility is reflected by considerable azimuthal disorder in radially-packed fibrils, with the azimuthal order specified, principally, in the cross-linked overlap zones; third, that mineral crystal size and order are tightly linked to the perfection of the collagen packing, although in bone the crystal c-axis may be tilted by as much as ±20° from the fibril axis; and fourth, that the process of mineralization may cause rearrangement of the cross-linkages in the collagen overlap zones.

Cranial sutures require tissue interactions with dura mater to resist osseous obliteration in vitro.

A chemically defined serum-free medium, which supports the development of bones and fibrous tissues of rat calvaria from nonmineralized mesenchymal precursor tissues, was employed to investigate tissue interactions between the dura matter and overlying tissues. Fetal calvarial rudiments from stages prior to bone and suture morphogenesis (fetal days 19 and 20) and neonatal calvarial rudiments with formed sutures (day 1) were cultured with and without associated dura mater. Removal of calvaria for in vitro culture allowed the examination of suture morphogenesis in the absence of tensional forces exerted on the sutures via fiber tracts in the dura mater originating in the cranial base. Ossification of frontal and parietal bones proceeded in a fashion comparable to development in vivo, but the cranial (coronal) sutures--primary sites for subsequent skull growth--were obliterated by osseous tissue union in the absence of dura mater. Bony fusion did not occur when rudiments were cocultured with dura mater on the opposite sides of 0.45 microns polycarbonate transwell filters, suggesting that the influence of dura mater on sutural obliteration was mediated by soluble factors rather than cell-cell or cell-matrix interactions. These results indicate that cell signaling mechanisms rather than biomechanical tensional forces are required for morphogenesis of the calvaria.

Localization and expression of osteopontin in mineralized and nonmineralized tissues of the periodontium.

To summarize results from various studies focusing on determining the expression/localization of BSP and OPN during tooth root development, there is general agreement that OPN is expressed/localized to the root surface during cementogenesis and is also seen throughout the PDL region. The expression/localization of OPN to odontoblasts and its role in dentinogenesis is less apparent. Recent studies directed at establishing odontoblast cell lines should help to resolve this conflict. Studies on BSP expression during tooth root formation indicate a very precise expression and localization of this molecule during cementogenesis indicating that this molecule may play an important role in the formation of this mineralized tissue. However, as with OPN, the expression of BSP and its role in dentin formation is not clearly defined.

Role of two mineral-associated adhesion molecules, osteopontin and bone sialoprotein, during cementogenesis.

Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21-42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non-mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis.

The Shanwang fossil biota in eastern China: a Miocene Konservat‐Lagerstätte in lacustrine deposits

The Miocene Shanwang biota in eastern China is one of the taxonomically most diversified and extraordinarily well-preserved lacustrine fossil deposits in the world. This study provides the historical and geological background, documents the taxonomic diversity, and examines the preservational style of this unique Konservar-Lagerstatte. A 30 m thick diatomaceous shale (the lower part of the Shanwang Formation), deposited in a small and volcanically related lake basin under a warm temperate climate, hosts more than 500 fossil species, including fungi, diatoms, higher plants, insects, ostracodes, fishes, amphibians, reptiles, birds, and mammals. The excellent preservation of the fossils has yielded (1) non-mineralized organic tissues of animals and plants; (2) detailed morphology of delicate organs such as flowers, feathers, and hairs; (3) original coloration of plants and insects; and (4) completely articulated animal bodies with in situ teeth, skin, and even stomach contents. The highly diversified fossil biota shows a complex interrelationship of co-occurring organisms that once lived in a eutrophic water body with a dense forest nearby. The preservation of soft tissues among the Shanwang fossils provides feasible material for further molecular-level investigation of these Miocene organisms. □Miocene, KONSERVAT-LAGERSTATTEN, lacustrine, taxonomic diversity, preservational style, Shanwang, China.

Digital image processing. II. In vitro quantitative evaluation of soft and hard peri-implant tissue changes.

The aim of this study was to evaluate the ability of computer-assisted densitometric image analysis (CADIA) to detect small changes in mineralized and nonmineralized tissues adjacent to dental implants and to correlate these changes with CADIA values. A section of a pig mandible including all soft tissues and in which a hollow cylinder ITI Bonefit implant with an artificial mesial and a buccal infrabony defect was placed was used to obtain pairs of standardized radiographs. Series of radiographs were obtained with exposure times of 0.13, 0.20, 0.44, and 0.53 s. Specimens of mineralized or nonmineralized tissues were placed arbitrarily in the defects before each radiographic exposure. The radiographs were captured through a video camera, digitized and stored in a personal computer. Every radiographic image was then subtracted from a baseline one without any change. The result of the subtraction was evaluated with CADIA. A linear correlation (r2 = 0.99) was found between the bone chips (1-5 mg of dry weight) placed in the mesial defect and the CADIA values. Bone chips in the buccal defect (behind the implant), however, were not detected unless their weight reached 14 mg or more. For conventionally exposed radiographs, it was not possible to recognize soft tissue specimens (1-6 mg), either in the buccal or the mesial defect. However, when "underexposed" radiographs (exposure time: 0.13 s) were obtained, a linear correlation (r2 = 0.80) was calculated for soft tissue specimens in the mesial defect and CADIA values. In normally exposed radiographs, the CADIA system could detect even the smallest change in bone density (bone chip of 1 mg of dry bone weight) and correlated almost linearly with these changes. Provided that the radiographic images are obtained with standardized geometry and normal exposure time, the tissue density changes detected by this system within bone defects represent only mineralized tissue changes. By underexposing radiographs, CADIA may even reveal soft tissue changes around dental implants.

Cloning and sequence analysis of bovine bone sialoprotein cDNA: conservation of acidic domains, tyrosine sulfation consensus repeats, and RGD cell attachment domain.

We isolated and sequenced a cDNA encoding bovine bone sialoprotein (BSP) using a bovine cDNA library made from mRNA isolated from bone-derived cell cultures and ligated to a phage lambda gt11. One of the cDNA clones isolated from this library had a 1800 base pair long insert and was found to contain the entire protein-encoding region. The deduced protein sequence revealed a 310 amino acid protein containing a signal peptide sequence of 16 hydrophobic amino acids. The protein sequence shows remarkable conservation with previously published human and rat sequences (more than 80% similarity for both species). The potential functional domains of BSP, including three acid amino acid-rich sequences, tyrosine sulfation consensus repeats, and the RGD cell binding sequence, are all present in the bovine sequence. Northern analysis of RNA from different bovine tissues indicated the presence of BSP message in bone but not in other nonmineralized tissues, confirming that bone is the major site of BSP message production.

Bone sialoprotein in developing porcine dental tissues: Cellular expression and comparison of tissue localization with osteopontin and osteonectin

Bone sialoprotein (BSP) is a highly sulphated and glycosylated phosphoprotein that is a major constituent of bone and other mineralized connective tissues. Although BSP can mediate cell attachment through an RGD sequence and binds selectively to hydroxyapatite, its precise function in mineralized tissues is unknown. To provide insights into its possible function, affinity-purified polyclonal antibodies directed against porcine BSP were used to demonstrate the histological distribution of this protein in developing porcine mandibular alveolar bone and the associated tooth tissues from 35- and 50-day fetuses. In addition, a porcine cRNA probe was used to determine the cellular expression of BSP in the same tissues by in situ hybridization. Immunoreactivity to BSP protein was restricted to the cells and matrix of the mineralized tissues of alveolar bone and dentine. In dentine, BSP was localized to the odontoblasts and their processes and to the peritubular dentine. In the alveolar bone, immunoreactivity for BSP was evident in osteoblastic cells and osteocytes and in the bone matrix; the older bone stained more strongly than newly formed bone. In addition, BSP appeared to be concentrated in the reversal lines of the rapidly remodelling bone. The distribution of BSP in these tissues revealed distinct differences when compared to osteopontin and SPARC/osteonectin, which are also prominent non-collagenous proteins of mineralized tissues. Most notable was the localization of osteopontin and especially osteonectin in non-mineralizing tissues. The immunoreactivity of osteoblasts and osteocytes for BSP in bone was consistent with the high levels of BSP mRNA revealed by in situ hybridization. However, much lower levels of hybridization were evident in the odontoblasts of developing mandibular molars. These studies demonstrate that BSP is expressed during the early formation of dentine and alveolar bone and that the protein accumulates in the peritubular dentine and bone matrix.