Ultrastructural immunolocalization of fibronectin in epiphyseal and metaphyseal bone of young rats

Fibronectin is a well known glycoprotein of extracellular connective tissue matrices due to a specific amino acid-sequence (RGD) suggested to act as an attachment factor in cell-cell or cell-matrix interactions. Although also present in bone, little is known about the role of fibronectin in this tissue. To obtain data for discussions on function we used ultrastructural immunolocalization techniques to quantitatively examine the distribution of fibronectin in various bone matrix compartments. The study was focused on three different stages of endochondral ossification in growing long bones of young rats. The results show large amounts of fibronectin in mature bone tissue. At a higher magnification, an obvious fibronectin association to individual fibrils of collagen type I was demonstrated. Intracellular labeling was observed in Golgi-related vesicles in some active osteoblasts of metaphyseal bone, indicating local synthesis of fibronectin. In contrast to previous suggestions based on light microscopic observations, the labeling of bone or cartilage matrices facing the surface of all cell types were low. The pattern is clearly different from that of osteopontin and bone sialoprotein, two other bone matrix proteins with the same cell-binding sequence. Our results indicate that fibronectin at these stages of development participates in matrix organization rather than being an important link between cartilage or bone matrix and adjacent cells.     

Nucleation of hydroxyapatite by bone sialoprotein

Bone sialoprotein (BSP) and osteopontin, the major phosphorylated proteins of mammalian bone, have been proposed to function in the initiation of mineralization. To test this hypothesis, the effects of BSP and osteopontin on hydroxyapatite crystal formation were determined by using a steady-state agarose gel system. At low calcium phosphate concentrations, no accumulation of calcium and phosphate occurred in control gels or gels containing osteopontin. Gels containing BSP at 1-5 micrograms/ml, however, exhibited a visible precipitation band and significantly elevated Ca + PO4 contents. By powder x-ray diffraction, the precipitate formed in the presence of BSP was shown to be hydroxyapatite. These findings suggest that bone sialoprotein may be involved in the nucleation of hydroxyapatite at the mineralization front of bone.     

Serum concentrations of cartilage oligomeric matrix protein and bone sialoprotein in hip osteoarthritis: a one year prospective study

OBJECTIVE: To evaluate serum concentrations of cartilage oligomeic matrix protein (COMP) and bone sialoprotein (BSP) as predictors of disease progression in hip osteoarthrtitis (OA). METHODS: Forty eight consecutive patients, referred to hospital for symptomatic hip OA, (ACR criteria) were monitored in a one year prospective trial with radiographs and serum samples. The radiographs were graded for joint space narrowing, osteophytes, and sclerosis and the joint space width was measured by a digitised image analyser. Serum COMP and BSP were quantified by immunoassays. RESULTS: The COMP concentrations at baseline correlated with the joint space width at entry and with its yearly mean narrowing (r = 0.38, p = 0.002) but not with joint space narrowing grade progression. The concentrations were higher in patients with bilateral hip OA (p = 0.03). The serum BSP concentrations at baseline were unrelated to OA progression but correlated inversely to the osteophyte grade (r = -0.36, p = 0.004) and sclerosis grade (r = -0.42, p = 0.0004). CONCLUSION: Serum COMP seems to be a surrogate marker of OA and may be of interest for the detection of patients at risk of rapidly progressing disease in hip OA. Serum BSP changes seem to reflect alterations in the subchondral bone turnover in hip OA. Measurement of joint space width using a digitised image analyser is a sensitive way of assessing OA progression that facilitates evaluation of tissue markers in relation to anatomical changes in the joint.     

Expression of rat bone sialoprotein promoter in transgenic mice

Bone sialoprotein (BSP) is a major protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite crystals. Our previous studies have demonstrated that BSP mRNA is expressed by differentiated osteoblasts, odontoblasts, and cementoblasts involved in de novo mineralized tissue formation in a tissue-specific and developmentally regulated manner. To determine the basis of the selective expression of the BSP gene, we have generated four transgenic mouse lines in which 2.7 kb of the rat BSP promoter ligated to a luciferase reporter gene has been stably integrated into the mouse genome. Assays of luciferase activities in 5-day-old animals has revealed consistently high levels in bone tissues with negligible activities in various other organs including kidney, liver, stomach, intestine, and spleen. In some animals, variable expression was observed in brain and skin. Temporal analyses revealed the highest luciferase expression in neonatal bones, with expression decreasing markedly with subsequent growth and development, as observed previously for the endogenous gene in rats. Immunohistochemical analysis of luciferase activity and in situ hybridization of luciferase mRNA in bone tissues show that differentiated osteoblasts express the highest levels of luciferase, consistent with the induction of endogenous gene expression. These studies demonstrate that the regulation of the BSP gene during osteoblastic differentiation, together with its tissue-specific, developmentally regulated expression, is primarily mediated within the 2.7 kb region of the promoter.     

Determination of the hydroxyapatite-nucleating region of bone sialoprotein

Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxyapatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3-5), two peptides of 38 kDa (P1) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133-272 (P1) and 42-125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, P1 and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3-5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA and BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA.     

Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.     

Periosteal new bone formation in a canine neuropathic model of osteoarthritis

OBJECTIVE: To characterize, for the first time, periosteal new bone formation in a well-established canine model of accelerated osteoarthritis (OA) with features of neuropathic arthropathy. METHODS: Seven dogs underwent left L4-S1 dorsal root ganglionectomy (DRG), followed 3 weeks later by transection of the anterior cruciate ligament of the ipsilateral knee (ACLT). Eight weeks thereafter, a postmortem examination was performed to assess the severity of cartilage changes of OA and the formation of new bone on the distal femur and proximal tibia in the cruciate-deficient limb. RESULTS: As described previously, extensive full-thickness ulceration of the articular cartilage was present in the unstable knee of every dog. The femoral shaft immediately proximal to the condyles in the unstable limb was consistently wider (mean +/- SD diameter 22.4 +/- 2.2 mm) than that in the contralateral limb (19.9 +/- 1.3 mm; P = 0.01). Xeroradiography and histologic examination of the distal femur revealed extensive formation of woven bone on the periosteal surfaces of the medial, lateral, and anterior aspects of the femoral shaft in the OA limb of every dog. These bony changes were not seen in radiographs of dogs that underwent DRG with the cruciate ligament left intact (n = 8) or of neurologically intact dogs that underwent ACLT (n = 7) and were examined 24 weeks after surgery. CONCLUSION: Formation of new periosteal bone on the distal femur and tibia is a feature of this model of accelerated OA that is not seen in the conventional ACLT model of OA in the neurologically intact dog. This observation suggests that interruption of sensory input from the limb may affect the regulation of osteogenesis in the mechanically unstable joint.     

IGF/IGFBP axis in cartilage and bone in osteoarthritis pathogenesis

In the context of joint biology, insulin-like growth factor-1 (IGF-1) is the most likely candidate to affect the anabolism of cartilage matrix molecules. Mechanisms for controlling the effects of IGF-1 include alterations in the level of this growth factor, its receptor and/or the IGF-1 affinity or availability to its receptor. Disturbance of any one of the above elements may induce a disregulation of the mechanisms involved in the local control of joint tissue integrity. This review focuses on recent studies of the IGF system, and the potential relevance of these results to in vivo effects in osteoarthritic (OA) tissues. It has been shown that, although the IGF-1's expression and synthesis are increased in OA cartilage, chondrocytes are hyporesponsive to IGF-1 stimulation. This phenomenon appears to be related, at least in part, to an increased level of IGF-binding proteins (IGFBP). The IGFBP have a high affinity for IGF-1, and appear to be important biomodulators for IGF action. Though to date seven IGFBP have been cloned and sequenced, disregulation in IGFBP-3 and -4 appears instrumental to arthritic disorders. Proteolytic activity directed against IGFBP has been found in both cartilage and bone; this activity appears to belong to serine- and/or metallo-proteases families. It has been suggested that a thickening of the subchondral bone participates in OA pathophysiology, and that IGF-1 production by bone and/or subchondral bone cells may contribute to these changes. An abnormal regulation of subchondral bone formation via an increase in the local activation of IGF-1 in bone cells, possibly via abnormal IGFBP synthesis due to aberrant PA/plasmin regulation of the IGF-I/IGFBP system, is believed to be a plausible hypothesis.     

Effect of age and osteoarthritis on bone mineral in rhesus monkey vertebrae

We have used vertebrae of free-ranging rhesus macaques to study the effect of age and osteoarthritis on bone mineralization and bone density and to relate these findings to weight, sex, parity, and mineral chemistry. Bone mineralization was determined using the density fractionation technique and bone density using dual-photon absorptiometry. Arthritis was determined osteologically. We found a relationship between mineralization, age, and osteophytes, such that mineralization rose with age in nonarthritics and decreased with age in arthritics. This could also be seen when the females were examined separately. In males, only an increase in mineralization with age could be seen. In females mineralization decreases with parity. Also in females, DPA density decreases with age and increases with parity. No relationships with DPA density could be seen using males and females together or males alone. In conclusion, we have shown that normal skeletal aging in rhesus monkeys is accompanied by an increase in mineralization similar to that in other species, but this is not true in the presence of osteoarthritis. In the females parity has an important effect because it seems to build up bone mass even though the bone present may be undermineralized.     

The spatial and temporal immunolocalization of TGF-beta 1 and bone morphogenetic protein-2/-4 in phallic bone formation in inbred Sprague Dawley male rats

Classical and artificial xenodiagnostic techniques made with Dipetalogaster maximus of first stage were performed simultaneously in 57 patients with chronic T. cruzi infection (22 male and 35 female patients, aged 7-80 years). With the exception of two patients with megaoesophagus, all had two previous positive serological reaction and a further test was done at the time of the examination. The patients came from the outpatient department of the university hospital or were resident in Mambaí, Goiás. Of the 57 patients, 24 (42%) had a positive xenodiagnoses. Of a total of 114 tests performed, 36(32%) were positive. Comparing the two xenodiagnostic techniques, no significant advantage was apparent statistically (p = 0.42), but the artificial technique has advantages because the blood is offered for triatomines through a device while in the classical technique, the triatomines suck through the patient's skin.     

The interaction of the zone of calcified cartilage and subchondral bone in osteoarthritis

The zone of calcified cartilage (ZCC) forms an important interface between cartilage and bone for transmitting force, attaching cartilage to bone, and limiting diffusion from bone to the deeper layers of cartilage. The height of the ZCC is a relatively constant percent of articular cartilage and the height is maintained by a balance between progression of the tidemark into the unmineralized cartilage and changing into bone by vascular invasion and bony remodeling. During its formation, the cells that form the ZCC have properties similar to the cells of the growth plate. In the adult, the ZCC becomes quiescent but not inactive. The ZCC may be reactivated in osteoarthritis and may progressively calcify the unmineralized cartilage. This might contribute to cartilage thinning which would increase the concentration of forces across the uncalcified cartilage leading to more damage. Although the subchondral bony plate remodels extensively in osteoarthritis, there is little evidence that a change in the biomechanics of the plate directly initiates the osteoarthritic process in cartilage. However, increased repair by endochondral ossification of vertical cracks in the ZCC that penetrate into the marrow space could contribute to progression via changes in the ZCC.     

Identification and immunolocalization of ecto-ATPDase in chicken stomach

The distribution of the extracellular adenosine tri- and di-phosphatase (ecto-ATPDase) in adult chicken tissues was investigated using a monoclonal antibody (MC18) generated previously from chicken oviduct. Ecto-ATPDase was determined to be most abundant in stomach by Western blot analysis of crude tissue homogenates. The ecto-ATPDase activity from solubilized stomach microsomes and a purified oviduct control was depleted 64% and 72%, respectively, by immunoprecipitation with MC18. Both oviduct and stomach ecto-ATPDases had an M(r) of approximately 80 kDa based on SDS-PAGE analysis. In addition, the enzymology of the ecto-ATPDase from both tissues was very similar. It is concluded that the same ecto-ATPDase is present in stomach and oviduct. Furthermore, immunolocalization of the stomach ecto-ATPDase with MC18 showed the enzyme to be localized in the apical membranes of the oxyntico-peptic cells, suggesting a role for the ecto-ATPDase in secretion.     

Ultrastructural and lanthanum tracer examination of rapidly resorbing rat alveolar bone suggests that osteoclasts internalize dying bone cells

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors have been shown to ameliorate the progression of glomerulosclerosis both in experimental models of uraemia and in patients with renal failure. It has not been documented, however, whether this is due to a decrease in angiotensin II generation or is a consequence of elevated local level of bradykinin. METHODS: Morphometric investigation of renal tissue was performed in 5/6 nephrectomized (SNx) rats, i.e. untreated or treated with the ACE inhibitor ramipril (SNx-RAM), the B2 kinin receptor antagonist HOE 140 (SNx-HOE), or a combination of both (SNx-RAM + HOE) over 8 weeks. A further group of SNx received delayed treatment with ramipril from week 5 onward (SNx-RAMD). In addition, a sham-operated (SHAM) control group was studied. RESULTS: Systolic blood pressure was significantly lower in both SNx-RAM and SNx-RAM + HOE groups compared to (untreated) SNx. The glomerulosclerosis index (GSI) was substantially higher in the (untreated) SNx group (0.24 +/- 0.04) vs SHAM (0.02 +/- 0.01). A significantly higher GSI was found in the SNx-HOE group (0.45 +/- 0.08) as compared to (untreated) SNx. However, in the SNx-RAM, SNx-RAM + HOE, and SNx-RAMD groups, the GSI was lowered to a similar extent (0.1 +/- 0.02, 0.09 +/- 0.02, and 0.07 +/- 0.01 respectively). In addition, a concomitant attenuation of tubulointerstitial damage was noted in all the above groups. CONCLUSION: Increased kinin activity does not appear to play a major role in the renoprotective effect of ACE inhibitors in the remnant kidney model.     

Ultrastructural study of the A-W GC-bone interface after long-term implantation in rat and human bone

The interface between apatite- and wollastonite-containing glass-ceramic (A-W GC) and bone after long-term implantation was studied by scanning and transmission electron microscopy (SEM and TEM) using rat and human specimens. First, particles of A-W GC (100-220 microns in diameter) were implanted into rat tibiae, and specimens were prepared for observation at 24, 48, 72, and 96 weeks after the operation. These long-term specimens showed an A-W GC-bone interface different from that at an earlier stage, which was investigated in our previous studies. SEM showed that the Ca-P-rich layer was wider, suggesting that leaching of ions from the A-W GC had continued even after bonding with bone. In some regions, the material particles were evidently replaced by the bone. TEM showed that the intervening apatite layer had become indistinct, and that A-W GC had intermingled with bone at the interface. In some regions, the surface of the A-W GC was degraded. These findings suggest that the surface region of A-W GC is slowly replaced by bone. Second, a human bone specimen, which included A-W GC particles (300-700 microns in diameter) implanted as a bone filler for about 75 weeks was harvested and investigated. Excellent A-W GC-bone bonding was observed, and the ultrastructure of the interface was similar to that in rats after long-term implantation. This finding demonstrated that A-W GC possibly worked in human bone in the same way as in rat bone, showing excellent bioactivity.     

Ultrastructural evaluation of the interaction of glucocorticoids and vitamin D on bone cells in thyroparathyroidectomized rats

To determine the direct effects of cortisol on bone, rats were thyroparathyroidectomized (T(X)PT(X)), fed a low-calcium diet, and given high (50 mg/kg) or low (8 mg/kg) pharmacologic levels of cortisol with or without excess vitamin D3 (15,000 IU). Rats given vitamin D had osteoblasts and osteocytes interpreted ultrastructurally to be actively engaged in matrix synthesis, mineralization of matrix, and in calcium mobilization. Osteoclasts were numerous on metaphyseal trabeculae and in vascular channels of cortical bone. In T(X)PT(X) rats not given vitamin D, osteoblasts and osteocytes were interpreted to have reduced metabolic activity with minimal evidence of participation in bone formation or resorption. Cortisol at both dose levels failed to alter the electron microscopic appearance of osteoblasts, osteocytes, and osteoclasts with or without vitamin D. Bone turnover indicated by urinary hydroxyproline excretion was unaffected by cortisol treatment. These findings suggest that glucocorticoids have little direct action on bone cells and that their effects on calcium metabolism are probably mediated by an interference in intestinal calcium transport and by secondary hyperparathyroidism.     

Characterization of native and recombinant bone sialoprotein: delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain

Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106-01 BSP) culture media is shown to have a hydroxyapatite Kd approximately 2.6 x 10(-9) M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR-BSP (Fragment 1 approximately 133A- approximately 265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post-translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral-binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a 15N-enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR-BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non-RGD cell adhesion sites are localized within conserved amino- and carboxy-terminal tyrosine-rich domains of recombinant human BSP. Given the proximity of the latter non-RGD cell adhesion site to the RGD tripeptide, a model of BSP-receptor interactions is presented.