The isolation and characterization of glycosylated phosphoproteins from herring fish bones.

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones.

The effects of high-dose, long-term alendronate treatment on microarchitecture and bone mineral density of compact and trabecular bone in the proximal femur of adult male rabbits.

Introduction: Despite the widespread use of bisphosphonates, its effects on normal bone microarchitecture of the proximal femur are still poorly studied. The purpose of this study was to determine the effects of long-term high-dose treatment of alendronate on microstructure and bone mineral density of cancellous, cortical compact and subchondral compact bone of the femoral head and neck region in normal adult male rabbits.

Materials And Methods: Thirty-two adult, male rabbits were randomized into and were treated with either alendronate or placebo for 6 and 12 months.

Bone matrix imaged in vivo by water- and fat-suppressed proton projection MRI (WASPI) of animal and human subjects.

Purpose: To demonstrate water- and fat-suppressed proton projection MRI (WASPI) in a clinical scanner to visualize the solid bone matrix in animal and human subjects.

Materials And Methods: Pig bone specimens and polymer pellets were used to optimize the WASPI method in terms of soft-tissue suppression, image resolution, signal-to-noise ratio, and scan time on a 3T MRI scanner. The ankles of healthy 2-3-month-old live Yorkshire pigs were scanned with the optimized method.

Quantitative bone matrix density measurement by water- and fat-suppressed proton projection MRI (WASPI) with polymer calibration phantoms.

The density of the organic matrix of bone substance is a critical parameter necessary to clinically evaluate and distinguish structural and metabolic pathological conditions such as osteomalacia in adults and rickets in growing children. Water- and fat-suppressed proton projection MRI (WASPI) was developed as a noninvasive means to obtain this information. In this study, a density calibration phantom was developed to convert WASPI intensity to true bone matrix density.

A comparison of the physical and chemical differences between cancellous and cortical bovine bone mineral at two ages.

To assess possible differences between the mineral phases of cortical and cancellous bone, the structure and composition of isolated bovine mineral crystals from young (1-3 months) and old (4-5 years) postnatal bovine animals were analyzed by a variety of complementary techniques: chemical analyses, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and (31)P solid-state magic angle spinning nuclear magnetic resonance spectroscopy (NMR). This combination of methods represents the most complete physicochemical characterization of cancellous and cortical bone mineral completed thus far. Spectra obtained from XRD, FTIR, and (31)P NMR all confirmed that the mineral was calcium phosphate in the form of carbonated apatite; however, a crystal maturation process was evident between the young and old and between cancellous and cortical mineral crystals.

Lateral packing of mineral crystals in bone collagen fibrils.

Combined small-angle x-ray scattering and transmission electron microscopy studies of intramuscular fish bone (shad and herring) indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, taking preferred orientation effects into account. Using the model, the diffuse fan-shaped small-angle x-ray scattering intensity profile, dominating the equatorial region of the scattering pattern, could be quantitatively analyzed as a function of the degree of mineralization.

Identification of osteopontin phosphorylation sites involved in bone remodeling and inhibition of pathological calcification.

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.

Age-dependent expression of VEGF isoforms and receptors in the rabbit anterior cruciate ligament.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF. Those isoforms differ in biochemical and biological properties, and it has been reported that their expression patterns are tissue and age specific as well. We investigated the expression levels of VEGF isoforms (VEGF121, VEGF165, VEGF183, VEGF189) and its receptors (VEGFR-1, flt-1 and VEGFR-2, flk-1/KDR) in the anterior cruciate ligament (ACL) of 2- to 3-week-, 2-month-, and 18-month-old New Zealand White rabbits using Sybr green Real-Time RT-PCR.

Changes in bone microarchitecture and bone mineral density following experimental osteonecrosis of the hip in rabbits.

Background: Osteonecrosis of the femoral head is a common disorder which can lead to hip joint destruction usually necessitating total hip replacement.

Methods: Quantitative micro-computed tomography, digital radiography and histology were used to characterize changes in bone microarchitecture and bone mineral density during the repair of the osteonecrotic femoral head as well as during the development of secondary osteoarthritis in the ipsilateral acetabulum. Osteonecrosis was induced surgically in 17 adult, male rabbits and the contralateral side was used as control.

Water- and fat-suppressed proton projection MRI (WASPI) of rat femur bone.

Investigators often study rats by microCT to investigate the pathogenesis and treatment of skeletal disorders in humans. However, microCT measurements provide information only on bone mineral content and not the solid matrix. CT scans are often carried out on cancellous bone, which contains a significant volume of marrow cells, stroma, water, and fat, and thus the apparent bone mineral density (BMD) does not reflect the mineral density within the matrix, where the mineral crystals are localized.

Site-specific in vivo calcification and osteogenesis stimulated by bone sialoprotein.

Bone sialoprotein (BSP) is one of the major non-collagenous glycosylated phosphoproteins of the extracellular matrix in bone. In vitro studies suggest that BSP may play important roles in the initiation and/or growth of calcium-phosphate crystals. To investigate the potential role of BSP in more complex in vivo environments, we implanted purified bovine BSP with type-I collagen as a carrier into surgically created rat calvarial defects and thoracic subcutaneous pouches.

Regulation of adult bone mass by the zinc finger adapter protein Schnurri-3.

Genetic mutations that disrupt osteoblast function can result in skeletal dysmorphogenesis or, more rarely, in increased postnatal bone formation. Here we show that Schnurri-3 (Shn3), a mammalian homolog of the Drosophila zinc finger adapter protein Shn, is an essential regulator of adult bone formation. Mice lacking Shn3 display adult-onset osteosclerosis with increased bone mass due to augmented osteoblast activity.

Prokaryotic expression of bone sialoprotein and identification of casein kinase II phosphorylation sites.

Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II.

Systemic hypoxia alters gene expression levels of structural proteins and growth factors in knee joint cartilage.

We investigated the effects of short- (8- and 24-h) and long-term (3 weeks) exposure to systemic normobaric hypoxia (13%) on the gene expression level of structural proteins and growth factors in knee joint cartilage of rabbits. Collagen type Ia2, II, and Va1, TGF-beta1, and b-FGF were upregulated after short-term hypoxia in both menisci, but not in articular cartilage. In contrast, long-term hypoxia downregulated gene expression level of collagens, aggrecan, and growth factors in articular cartilage and meniscal fibrocartilage.

Differential expression of VEGF isoforms and receptors in knee joint menisci under systemic hypoxia.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. We examined the expression levels of VEGF isoforms and their receptors in the medial and lateral meniscus of rabbits under normal physiologic conditions as well their expression levels after 8 and 24 h of systemic normobaric hypoxia (13%). VEGF121 is the most abundant VEGF isoform in the medial and lateral meniscus, followed by VEGF165, VEGF189, and VEGF183.

Poorly crystalline apatites: evolution and maturation in vitro and in vivo.

Poorly crystalline apatites (PCA) are the major mineral component of mineralized tissues in vertebrates. Their physical-chemical properties are, however, not very well known due to their relative instability and the difficulties to characterize nanocrystalline compounds. Several studies using spectroscopic techniques (Fourier transform infrared [FTIR]; 31P nuclear magnetic resonance [NMR]) have demonstrated the existence, both in precipitated and biological PCA, of labile non-apatitic environments of the mineral ions.

Absence of transcription factor c-maf causes abnormal terminal differentiation of hypertrophic chondrocytes during endochondral bone development.

In this study, we report that the transcription factor c-Maf is required for normal chondrocyte differentiation during endochondral bone development. c-maf is expressed in hypertrophic chondrocytes during fetal development (E14.5-E18.

Density of organic matrix of native mineralized bone measured by water- and fat-suppressed proton projection MRI.

Water- and fat-suppressed projection MR imaging (WASPI) utilizes the large difference between the proton T(2) (*)s of the solid organic matrix and the fluid constituents of bone to suppress the fluid signals while preserving solid matrix signals. The solid constituents include collagen and some molecularly immobile water and exhibit very short T(2) (*). The fluid constituents include mobile water and fat, with long T(2) (*).

Hydroxyapatite crystals as a local delivery system for cisplatin: adsorption and release of cisplatin in vitro.

This study describes the characteristics of the in vitro binding and release of the anti-tumor drug cisplatin by slurries of synthetic hydroxyapatite crystals carried out in aqueous media. The adsorption of cisplatin by slurries of hydroxyapatite and its release were found to depend significantly on the ionic composition of the aqueous media used. At a constant pH of 7.

Nuclear magnetic resonance spin-spin relaxation of the crystals of bone, dental enamel, and synthetic hydroxyapatites.

Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short-range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus-31 solid state nuclear magnetic resonance (NMR) spin-spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained from pure and carbonated hydroxyapatites.