Further evidence linking urolithiasis and blood coagulation: urinary prothrombin fragment 1 is present in stone matrix

The fact that organic material is always present and distributed throughout each renal calculus suggests that it may play a role in stone formation. The organic matrix of calcium oxalate (CaOx) crystals freshly generated in urine in vitro contains urinary prothrombin fragment 1 (UPTF1) as the principal protein. In this initial study, matrix was extracted from 12 renal calculi and evaluated for the presence of UPTF1 using Western blotting. UPTF1 was present in all eight stones whose principal component was CaOx, and in one of two stones which consisted mainly of calcium phosphate (CaP). UPTF1 was absent from the two struvite calculi examined. The relationship between CaP and UPTF1 was explored further. Matrix harvested from CaP crystals freshly generated in urine in vitro was also shown to contain UPTF1 as its principal component. Our inability to detect UPTF1 in one mixed CaOx/CaP stone may be related to our methods of matrix retrieval, while its absence from two struvite stones argues against it being present in the other stones merely as a consequence of passive inclusion. This absence may be related to the alkaline environment typical of struvite stone growth. The finding that UPTF1 is present in some renal stones provides the first direct evidence that links blood coagulation proteins with urolithiasis.     

Urinary stone proteins: an update

The discovery of an organic component in kidney stones dates back to 1684. More than 150 years elapsed before the incrustation of this organic component, which is now called the matrix, was proposed as the mechanism of stone formation. The composition of the matrix remained largely unknown until the development of electron microscopy and the advances in biochemistry combined in the 1950's to usher in the modern era of renal stone matrix investigation. Composed mainly of selectively incorporated proteins generally characterized by high glutamic and aspartic acid content and the frequent occurrence of gamma-carboxyglutamic acid, the matrix displays a variable and complex composition and shares a few proteins in many stones. The embryonic stone may first appear in the renal tubules where it can acquire the blood and cell membrane proteins recently found by analysis of stone protein extracts. The combination of supersaturation, an appropriate environment, the availability of calcium binding proteins which may be abnormal, and the incorporation of proteins extracted from leukocytes and cell wall membranes may induce stone formation.     

Adhesion and endocytosis of calcium oxalate crystals on renal tubular cells

The present investigation was designed to study interactions between Madin-Darby canine kidney (MDCK) cells and calcium oxalate monohydrate (COM) crystals and to clarify the significance of these crystal-cell interactions in stone pathogenesis. MDCK cells cultured in the presence of COM crystals showed a time-dependent uptake of crystals; this was specific for COM crystals. In the dynamic model system designed to study these phenomena under more physiological conditions, COM crystals adhered to the cell surface and were subsequently internalized. In this endocytotic process, the microvilli of the cell appeared to play an important role. The observation by scanning electron microscopy of complexes consisting of aggregated COM crystals and cell debris led us to speculate that adhesion and endocytosis of crystals might provide the calculus nidus for aggregation and retention of crystals in the renal tubule. Furthermore, glycosaminoglycans and the macromolecular fraction of human urine were shown to have the ability to inhibit the cellular uptake of crystals. Evidence that similar processes may also occur in vivo was obtained using an experimental stone model in rats. Our experiments revealed that most of the COM crystals adhered to the tubular cells and some crystals were endocytosed by the cell. Thus, these crystal-cell interactions might be one of the earliest processes in the formation of kidney stones. Further elucidation of the mechanism and the regulatory factors involved in this process may provide new insight into stone pathogenesis.     

Effect of lactic acid and proteolytic enzymes on the release of organic matrix components from human root dentin

The mechanisms of organic matrix breakdown in the root caries process are not well understood. Therefore, the combined and separate effects of lactic acid and proteolytic enzymes on the degradation of human dentin collagen, glycoproteins, proteoglycans and phosphoproteins were investigated in the present study. Dentin powder was pretreated with lactic acid (pH 4.0), distilled and deionized (dd) water (pH 7.0) and EDTA/guanidine HCl (pH 7.4) for 24 h. Pellets of acid- or dd water-pretreated dentin powder were washed, dried, and then treated with trypsin, bacterial or mammalian tissue collagenase, or control buffer for 3 h. The released dentin proteins were analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify degraded type I collagen, proteoglycans, glycoproteins and phosphoproteins. All water and acid pretreatment and enzyme treatment groups demonstrated two collagen fragment bands with molecular weights at approximately 79 kD. Further studies showed that the 79 kD proteins from acid-pretreated dentin collagen were degraded by tissue collagenase, suggesting that endogenous collagenase may be involved in the degradation of root dentin collagen. Dentin proteoglycans were detectable in all the treatment groups by protein slot blotting. Relatively few distinct glycoproteins and proteoglycans, and no phosphoproteins were detected by immunoblotting. Results from this study suggest that both acids and proteolytic enzymes from either host or microbial origin are important in the degradation of human dentin matrix and the mechanisms involved in the release of various noncollagenous proteins may be different.     

Uropontin in urinary calcium stone formation

Normal urine is frequently supersaturated with respect to calcium oxalate. Thus, urinary inhibitors of crystallization appear to have an important role in preventing urinary stone formation. Uropontin was isolated by monoclonal antibody immunoaffinity chromatography and has the same N-terminal sequence as osteopontin derived from bone. This urinary form of osteopontin is a potent inhibitor of calcium oxalate monohydrate crystal growth at concentrations (approximately 0.1 microM) that normally prevail in human urine. Interaction with calcium oxalate monohydrate in vivo was shown by analysis of EDTA extracts of calcium stones. Uropontin is an abundant component of calcium oxalate monohydrate stones and present in only trace quantities in calcium oxalate dihydrate and hydroxyapatite stones. However, the precise role of uropontin in the pathogenesis of urinary stone formation is not known and is the subject of ongoing investigations.     

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori

Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG1, IgG3 and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.     

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.     

Localization of estrogen-receptor-related antigen in human odontoblasts

Estrogen receptors have been demonstrated in many osteogenic cell lines. Recently, we showed that estrogen deficiency induced by ovariectomy caused enhanced dentin formation in adult rats, suggesting that estrogen receptors may be present in dental tissues. Nothing is known about estrogen receptors in human teeth. We used immunohistochemical staining and immuno-blotting to demonstrate the presence of estrogen receptors in human pulp and/or the pulpo-dentinal border. Unerupted human wisdom teeth were surgically removed, frozen in liquid nitrogen, and prepared for immunological studies. Western blot analysis with monoclonal antibodies specific for human estrogen-receptor-related antigens demonstrated an approximately 29-kDa clear double band in the material scraped from the predentin-odontoblast border and in the fluid that emerged into the pulpal chamber, evidently from the odontoblasts. A weaker double band was also present in pulpal tissue samples. By immunohistochemical staining, estrogen-receptor-related antigens were visualized in the predentinal-odontoblast region and in the pulpal blood vessels. Our results suggest the presence of estrogen receptors in human teeth, and thus the previously reported enhancement of the dentin formation in rats after ovariectomy may be mediated via these receptors.     

In vitro modeling of the bone/implant interface

BACKGROUND: The purpose of this review is to examine the usefulness of cell culture methods to model the mechanisms of bone formation on the surfaces of candidate implant materials. METHODS: The central objective is to show that in vitro methods are uniquely valuable in providing an understanding of how new bone is formed on solid surfaces. It should be emphasized, at the outset, that the use of cell culture studies as cytotoxicity assays will not be addressed, nor is it implied that cell cultures can model all the complexities of the in vivo environment. Nevertheless, by comparison with in vivo data, which are by nature retrospective, it is shown that primary differentiating osteogenic cell cultures, derived from bone marrow, illustrate a sequence of extracellular matrix elaboration events that characterize the establishment of the interface between newly formed bone and solid surfaces. These solid surfaces either may be implant materials, or indeed previously formed bone matrix, which has been resorbed during normal bone remodeling events. In each case the first biologically derived matrix at these sites is a morphologically distinct collagen fibre-free extracellular matrix, which, in bone histology has been referred to for > 100 years as a cement line. RESULTS: The sequence starts with secretion and adsorption to the substratum of organic components, of which the major proteins are osteopontin and bone sialoprotein. Mineralization of this matrix occurs by the seeding of nanocrystalline calcium phosphate, which precedes the appearance of morphologically identifiable collagen fibres. This is clearly contrary to the dogma that collagen is necessary for mineralization of bone, but is in agreement with specific cases of other, particularly dental, calcified connective tissues. Although collagen is synthesized by the differentiating osteogenic cells that elaborate the cement line interface, it is not adsorbed to the underlying solid surface. Following the elaboration of the cement line matrix, collagen fibre assembly occurs and is then mineralized to produce morphologically identifiable bone matrix. CONCLUSION: Key elements of this sequence of events can be seen at the interface of implants retrieved from in vivo experiments, which indicates that these in vitro methods not only mimic known in vivo phenomena, but also provide a mechanistic understanding of bone elaboration at implant surfaces. However, distinction is drawn between the events of new bone formation at implant surfaces and other bone/implant morphologies, which are unrelated to de novo bone formation at the implant surface. Finally, this new information emerging from bone marrow cell culture studies demands a re-examination of the concepts of bone-bonding and nonbonding implant materials.     

Edgard C. Moreno, PhD: a tribute to his major contributions to oral biology research

The seminal contributions of Dr. Edgard C. Moreno to physical chemical aspects of oral biology research are reviewed, with personal insights provided by the author. Dr. Moreno has made major contributions to our understanding of fundamental processes which occur within the oral cavity and which control the formation of mineralized tissues. These contributions include extensive research in the areas of: the chemistry of synthetic calcium phosphates and biominerals, the interaction of salivary proteins and other molecules of biological interest with calcium phosphate surfaces, the mechanism of dental caries formation and the effects of fluoride on this process, the mechanism of dental calculus formation and prevention, and the mechanism of dental enamel formation.     

Fluoride profiles in dental calculus from Japanese, Chinese and British residents

Whether the fluoride concentrations and profiles differ in human dental calculus obtained from different countries was investigated. A total of 203 dental calculus deposits on 203 permanent teeth from residents (mean age, 52.1 years) of Nagoya (Japan), Shanghai (China), Leeds (Great Britain) and the Wuhan mountainous area (China, fluoridated area) were analysed. An abrasive microsampling procedure was used to examine fluoride distribution. There were five types of fluoride profiles in dental calculus in each area/country (designated types L, J, U, T, W). In supragingival calculus, type L (highest in the outermost layers) and type J (highest in the innermost layers) both had significantly higher values than type U (high in the surface and innermost layers) but were relatively identical. In subgingival calculus, type W (high in the outermost, mid and innermost layers) was characteristics. Calculus from the Wuhan mountainous area (fluoridated) had the highest fluoride concentration, followed by Leeds (non-fluoridated), and Nagoya and Shanghai (non-fluoridated) calculus had the lowest. Fluoride concentrations in supragingival calculus were higher in teeth extracted because of periodontal diseases than dental caries. It is concluded that fluoride concentrations and distribution in dental calculus differ from country to country, probably due to different fluoride environments.     

C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries

There is increasing evidence that complement activation may play a role in atherogenesis. Complement proteins have been demonstrated to be present in early atherosclerotic lesions of animals and humans, and cholesterol-induced atherosclerotic lesion formation is reduced in complement-deficient animals. Potential complement activators in atherosclerotic lesions are now a subject matter of debate. C-reactive protein (CRP) is an acute-phase protein that is involved in inflammatory processes in numerous ways. It binds to lipoproteins and activates the complement system via the classic pathway. In this study we have investigated early atherosclerotic lesions of human coronary arteries by means of immunohistochemical staining. We demonstrate here that CRP deposits in the arterial wall in early atherosclerotic lesions with 2 predominant manifestations. First, there is a diffuse rather than a focal deposition in the deep fibroelastic layer and in the fibromuscular layer of the intima adjacent to the media. In this location, CRP frequently colocalizes with the terminal complement complex. Second, the majority of foam cells below the endothelium show positive staining for CRP. In this location, no colocalization with the terminal complement proteins can be observed. Our data suggest that CRP may promote atherosclerotic lesion formation by activating the complement system and being involved in foam cell formation.     

Recombinant human osteogenic protein-1 induces dentin formation: an experimental study in miniature swine

It was the aim of the present study to investigate the induction of dentin formation by recombinant human osteogenic protein-1 (hOP-1). In 4 adult miniature pigs a total of 16 teeth with artificially exposed dental pulps were capped with 3 mg of a complex of recombinant hOP-1 in collagen matrix (2.5 micrograms/mg), collagen matrix alone, or calcium hydroxide paste. Teeth were removed in block section after a healing period of 5 weeks. Decalcified sections were processed for light microscopy and histomorphometric analysis. In hOP-1 treated teeth substantial amounts of hard tissue formation (osteodentin and tubular dentin) had consistently led to a complete bridging of the defects. Less dentin formation was seen after calcium hydroxide application. In control defects collagen matrix alone failed to form complete dentin bridges. Recombinant human osteogenic protein-1 in a collagen carrier matrix appeared to be suitable as a bio-active capping agent for surgically exposed dental pulps.     

Calculus deposits and bone loss on the teeth of Romano-British and eighteenth-century Londoners

The relation between dental calculus and periodontal disease is not clear but it is generally recognized that calculus is a significant pathogenetic factor. Skeletal material has previously been used to study some aspects of chronic adult periodontitis but few studies have quantified the extent of calculus in ancient populations and its relation to changes in alveolar bone height. This study records the presence and extent of calculus and its relation to alveolar bone loss in a Romano-British and eighteenth-century London population. There were significant differences in calculus deposition in the two populations but this appeared to have little effect on changes in alveolar bone contour. It is suggested that the amount of calculus may be related to diet but that changes in alveolar bone height seem to be independently controlled.     

Isoforms and levels of transferrin, antithrombin, alpha(1)-antitrypsin and thyroxine-binding globulin in 48 patients with carbohydrate-deficient glycoprotein syndrome type I

To improve our understanding of the role of extracellular matrix (ECM) proteins and integrins during the processes of granuloma formation in sarcoidosis, we examined the distribution of ECM proteins and the expression of integrins in sarcoid lymph nodes by immunohistochemical methods. We also examined the expression of transforming growth factor-beta1 (TGF-beta1), which is one of major regulators for synthesis of ECM proteins. Most ECM proteins were detected in the periphery of the granulomas in a concentric pattern, and fibronectin was diffusely detected from an early to a regressive stage. Compared with normal lymph nodes, most beta1-integrin subfamilies (alpha1, alpha4, alpha5 and alpha6) were more strongly expressed on lymphocytes around the granulomas. Epithelioid cells exhibited strong expression of the alpha5 molecule. Fibroblasts exhibited the expression of the alpha2 and alpha5 molecules surrounding ECM proteins. The alpha5beta1 molecule had a distribution similar to that of fibronectin. TGF-beta1 was detected in epithelioid cells throughout the various evolutional stages and its expression was especially marked in mature granulomas. Interaction of fibronectin and the alpha5beta1 molecule may have an important role in the process of formation of sarcoid granuloma. The expression of TGF-beta1 may be involved in the regression of sarcoid granuloma by initiating fibrosis and atrophy of epithelioid cells.     

The relationship of function and pathology of submandibular gland with sialolithiasis

Submandibular glands with sialolithiasis are analysed histopathologically. The formation of the calculi of the submandibular glands may be related to the sialoadentitis and to the changes of saliva composition. Mineralization of the organic matrix may be one of the cause of calculus formation. By comparison of the glandular function with the pathology of the gland, it shows that the reduction of glandular function is associated with the severity of grandular inflammation. When function index is normal, grandular inflammation is in the Stage I or II, while it decreases or zero, grandular inflammation is in the Stage III or IV. Glandular function index may predict the severity of grandular inflammation.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5-30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma.     

Extracellular calcium-binding proteins

Point mutations in Ca2+-binding sites of extracellular matrix proteins have been identified as the cause of human disorders such as Marfansyndrome and pseudoachondroplasia. Although the modes of Ca2+ binding and the effects of point mutations are not yet understood in these two cases, new insight was recently gained by X-ray and NMR structure determinations of several other extracellular proteins; these studies revealed a diversity of functions of Ca2+ ions. Ca2+ may induce a profound conformational change within a single domain, may bridge adjacent domains and thus direct the relative domain orientation and supramolecular structure, or may be involved in carbohydrate and membrane binding.