Marie Yamada

Morphological examination of bone synthesis via direct administration of basic fibroblast growth factor into rat bone marrow

Woven bone induced by direct injection of basic fibroblast growth factor (bFGF) into rat bone marrow was examined. On the first day after injection, fibrous tissues formed in the treated region of the bone marrow. Tissue‐nonspecific alkaline phosphatase (TNAPase)‐immunopositive osteoblastic cells and osteopontin immunopositive‐extracellular matrices were observed in the fibrous tissues, indicating bone induction. On the fifth day, the bFGF‐induced bone was found broadly in the bone marrow. In the originally existing bone, osteopontin‐immunoreactivity was observed at cement lines, but not in the fully calcified matrix, whereas the woven bone displayed immunoreactivity throughout the matrix. Numerous TRAPase‐positive osteoclasts were present on the surfaces of the woven bone, but no obvious cement line was observed. Therefore, both bone formation and resorption appeared highly active, without normal cellular coupling equilibrated between bone formation and resorption performed by osteoblasts and osteoclasts. On the tenth day, the bFGF‐induced bone was almost replaced by bone marrow. Thus, bone formation actively occurred in the first half of the experimental period, whereas bone resorption came to be predominant thereafter. This study demonstrated that bFGF stimulates bone formation, which, however, is subject to subsequent resorption, probably due in part to the absence of coordinated cellular coupling between osteoclasts and osteoblasts. Microsc. Res. Tech. 41:313–322, 1998.

Action of metalloproteinases on porcine dentin mineralization

Samples containing predentin and mineralized dentin involving the mineralized front (newly formed dentin) were prepared by scraping developing porcine teeth after odontoblastic cell debris had been removed from the predentin surfaces. An extract was obtained separately from the matrices of predentin and of the newly formed dentin with a 4 M guanidine solution before and after demineralization with acetic acid solution. Enzymography detected 56 and 61 kDa gelatinases and 25 kDa proteoglycanase as neutral metalloproteinases in both extracts and proved them to be in an active form. Approximately half of the 56 and 61 kDa gelaunases binds to collagen fibers in predentin matrix. Three high molecular weight proteoglycans (70–85 kDa, 130–180 kDa, and 290 kDa) were found in the predentin matrix, but not in the newly formed dentin. The proteoglycanases in predentin degraded 290 kDa proteoglycan, if incubated together with calcium (Ca) ions. The results of this investigation indicate that active proteoglycanases with existed in the predentin perform no substantial work in proteoglycan degradation because the Ca ions are masked in the predentin matrix by coexisting proteoglycans. When mineralization occurs, however, they can degrade the proteoglycan at the mineralization front because excess Ca ions may be supplied via odontoblastic processes.

Immunoblot detection and expression of enamel proteins at the apical portion of the forming root in porcine permanent incisor tooth germs.

Abstract There have been many immunohistochemical studies of enamel proteins during root formation. In the present article, the detection and expression of enamel proteins in tissue samples prepared from the apical portion of the forming root (APFR) in porcine permanent incisor tooth germs were studied. Amelogenin, enamelin, and sheathlin were detected by immunoblot analysis, but only in small amounts. The detection of their derivatives indicated their degradation. It is, at present, unclear as to which proteinases are involved in these degradations, because activity of enamel matrix serine proteinase 1 and enamelysin was not detected on gelatin and casein zymograms. The expression of enamel proteins was also proved in the APFR sample by the detection of polymerase chain reaction products of their cDNAs, and this may be related to cells of fragmentized Hertwigs epithelial root sheath. Amelogenin expression was not greater than that of enamelin and sheathlin. It was different from the expression pattern of secretory ameloblasts involved in enamel matrix formation. These results suggest that the amelogenins found in the APFR do not form a three-dimensional structure of amelogenin micelles, which has been proposed for the secretory enamel matrix structure. In this case, the enamel proteins could spread out easily following degradation into the matrix of future cementum. Some of their derivatives may play a role in the formation of the cementum.

The localization of CD44 and moesin in osteoclasts after calcitonin administration in mouse tibiae

We investigated the immunohistochemical localization of CD44, hyaluronate receptor, and moesin, of the ezrinradixin-moesin (ERM) family, in osteoclasts after calcitonin adminstration using confocal laser scanning microscopy and transmission electron microscopy to clarify the role of CD44 and moesin in their cytoskeletal organization and cell polarity. We also elucidated the localization of osteopontin (OPN) to confirm its possible role in cell-matrix recognition via CD44. In untreated mice, intense immunoreactivities for CD44 and moesin were detected on the basolateral plasma membrane of osteoclasts. Rhodamine-phalloidin reactivity was seen in a bandlike pattern on the region of contact between osteoclasts and bone and was also detected moderately along their basolateral plasma membrane. At 30 min after calcitonin administration, osteoclasts did not show either clear zones or ruffled borders. The bandlike reactivity of rhodamine-phalloidin in the contact region was diminished, although labeling was seen along osteoclasts. CD44 and moesin were colocalized along their plasma membranes, including the region facing the bone surface. Electron microscopic observation revealed that the microvillus processes in the contacting region with bone surface, as well as the basolateral plasma membrane, showed immunoreactivities to CD44 and moesin. At 60 min, some osteoclasts attached to bone and showed a bandlike pattern of rhodamine-phalloidin. On the other hand, OPN was localized under CD44-positive cytoplasmic processes and the clear zone of osteoclasts. These findings suggest that calcitonin effects on the cell polarity of osteoclasts and the CD44-moesin-actin filament system in osteoclasts plays an imporant role in cell polarity and cell-matrix recognition.

An active neutral metalloproteinase bound to the insoluble collagen in the mineralized phase matrix of adult rat calvaria

SummaryTwo kinds of proteinases were found in the mineralized phase matrix of 24-week-old rat calvaria by means of enzymography using gelatin as a substrate. One proteinase was a neutral thiol 58kD proteinase as shown in a previous paper [2]. The other was a neutral metalloproteinase that had a molecular mass of 56kD and was detected only when calcium (Ca) ions were added to the incubation buffer. It is believed that the 56kD proteinase is bound to the insoluble collagen of the bone matrix, as it is solubilized by 4 M guanidine HCl solution from the insoluble collagen fraction, when prepared by removing extractable proteins of the mineralized phase matrix. The insoluble collagen fraction could also be solubilized and prepared as gelatin by heating at 65°C for 5 minutes. The gelatin was then incubated at 37°C without further treatment and became degraded without an activation of 4-aminophenylmercuric acetate (APMA). This nonactivated degradation was enhanced by adding Ca ions. These results suggest that the 56kD metalloproteinase bound to the insoluble collagen of bone matrix is in an active form and may participate in the rapid degradation of collagen during bone resorption. As partially purified 56kD metalloproteinase degraded cartilage type proteoglycan, but not type I, IV, and V collagens, it is possibly related to the degradation of proteoglycans before it binds to collagen fibers during bone formation.

Noncollagenous proteins of newborn rat calvaria: the possible mineral and collagen binding proteins

In order to separate only the mineral and collagen binding (MCB) proteins from the mineral compartment of newborn rat calvaria after the mineral unassociated proteins were removed by washing in 4 M guanidine solution, the sequential extractions were carried out by homogenizing in acetic acid and several neutral solutions with a different ion concentration.Three noncollagenous proteins were solubilized as MCB proteins by sequential extractions and each had an affinity for calcium ions. One is the sialoprotein which has an apparent molecular weight (Mr=) of 80,000–115,000 and stains blue with Stains-all but does not stain with Coomassie blue (CBB), this sialoprotein is expected to bind more tightly to the matrix by ionic bonds than the other two proteins. The protein species of Mr=66,000 is a phosphorylated glycoprotein which contains a small amount of sialic acid and appears to bind to the matrix. Partial amino acid sequence analysis suggests that this protein is osteopontin reported by Oldberg et al (Proc. Natl. Acad. Sci. USA. 83, 8819–8823, 1986).The other Mr=49,000 species may have an affinity for the matrix, although part of this protein binds only to the mineral, and by N-terminal amino acid sequence analysis is found to be derived from serum and the analogue of humanα2HS glycoprotein.

Bone phosphoprotein of newborn rat calvaria detected histochemically with the cationic carbocyanine dye “Stains-all”

AbstructThe originally mineralized area of a fixed and demineralized specimen of newborn rat calvarium was stained blue with Stains-all, which stained phosphoprotiens, glycoproteins and Ca-binding proteins blue. The material stained blue in the histological sections was solubilized by an acidic solution and found to be the protein (Stains-all positive protein = SAPP). This SAPP was found to consist of two different types of phosphoproteins, which had the same amino acid sequence at the N-terminal region and a quite similar amino acid composition to each other. The minor one was equal to the 44kD phosphoprotein which has previously been reported by Prince et al (J. Biol. Chem., 262, 2900, 1987). The major one was a 66kD phosphoprotein, its molecular weight being determined by SDS electrophoresis on a 15% polyacrylamide gel. The result of the amino acid analysis and peptide mapping analysis after CNBr cleavage contained no Met. Based on its solubility characteristics at sequential extractions with three acidic solutions (pH 5.5, pH 3.5 and pH 2.6), the histochemically discovered SAPP was believed to be bound to the matrix by a weak ionic bond and covered with mineral. It is suggested that the Met free phosphoprotein is a new protein in the mineralized phase of newborn rat calvaria and participates in the induction of bone mineralization.

Rat incisor dentin formed under low plasma calcium concentration: Relation of phosphophoryns to the affected dentin

Abstract: When parathyroidectomized (PTX) rats were maintained on a diet containing 0.3% calcium and lacking vitamin D, the forming incisor dentin consisted of two distinct layers that differed in their degree of mineralization. The two layers could be distinguished by hematoxylin staining and by contact microradiography. The layer near the dental pulp was the hypomineralized dentin, different from a predentin, containing a small amount of mineral detected by electron probe and chemical analyses. The mineralization of the outer dentin was almost normal. Biochemical characterization demonstrated that the two dentin layers differed in their phosphophoryn content. The outer dentin contained two phosphophoryns, which is the pattern found in normal dentin. In contrast, the hypomineralized dentin contained only the lower molecular weight and lower phosphorylated phosphophoryn, which may be involved in crystal nucleation. From the distribution of lead, which marked dentin formed at the time of injection, it could be inferred that the mineralized dentin begins as a hypomineralized layer that undergoes secondary mineralization. The secondary mineralization occurs by the active calcium transport of the odontoblasts via odontoblastic processes.