Tomomaya Yamamoto

Twisted plywood structure of an alternating lamellar pattern in cellular cementum of human teeth

Human cellular cementum was examined by scanning electron microscopy to elucidate the manner of the alternate lamellar pattern forming the cellular cementum. Specimens were demineralized, trimmed with a freezing microtome, and treated by NaOH-maceration. This procedure was chosen to avoid artifacts in the fibril arrangement, and to study the fibrous architecture in detail. For comparison, non-demineralized, polished and HCl-etched specimens were also prepared. In the NaOH-macerated specimens, the lamellar pattern of the cellular cementum conformed to the twisted plywood principle of bone lamellation with a periodic rotation of matrix fibrils resulting in an alternating lamellar pattern. In contrast, matrix fibrils were irregularly arranged without indication of rotation of matrix fibrils in the polished and etched specimens. Our results suggest that polishing and etching procedures cause damage to fibrils and fibril arrangement.

Altered distribution of bone matrix proteins and defective bone mineralization in klotho-deficient mice

In an attempt to identify the histological properties of the klotho-deficient (kl/kl) bone matrix, bone mineralization and the localization of Ca(2+)-binding bone matrix proteins - osteocalcin, dentin matrix protein-1 (DMP-1) and matrix Gla protein (MGP) - were examined in kl/kl tibiae. While a widespread osteocalcin staining could be verified in the wild-type bone matrix, localization of the same protein in the kl/kl tibiae seemed rather restricted to osteocytes with only a faint staining of the whole bone matrix. In wild-type mice, MGP immunoreactivity was present at the junction between the epiphyseal bone and cartilage, and at the insertion of the cruciate ligaments. In kl/kl mice, however, MGP was seen around the cartilaginous cores of the metaphyseal trabeculae and in the periphery of some cells of the bone surface. DMP-1 was identified in the osteocytic canalicular system of wild-type tibiae, but in the kl/kl tibiae this protein was mostly found in the osteocytic lacunae and in the periphery of some cells of the bone surface. Mineralization of the kl/kl bone seemed somewhat defective, with broad unmineralized areas within its matrix. In these areas, mineralized osteocytes along with their lacunae and osteocytic cytoplasmic processes were found to have intense osteocalcin and DMP-1 staining. Taken together, it might be that the excessive production of Ca(2+)-binding molecules such as osteocalcin and DMP-1 by osteocytes concentrates mineralization around such cells, disturbing the completeness of mineralization in the kl/kl bone matrix.

Frequency of Teriparatide Administration Affects the Histological Pattern of Bone Formation in Young Adult Male Mice

Evidence supports that daily and once-weekly administration of teriparatide, human (h)PTH(1-34), enhance bone mass in osteoporotic patients. However, it is uncertain whether different frequencies of hPTH(1-34) administration would induce bone formation similarly in terms of quantity and quality. To investigate that issue, mice were subjected to different frequencies of PTH administration, and their bones were histologically examined. Frequencies of administration were 1 time/2 days, 1 time a day, and 2 and 4 times a day. Mice were allocated to either to control or to 3 different dosing regimens: 80 μg/kg of hPTH(1-34) per injection (80 μg/kg per dose), 80 μg/kg of hPTH(1-34) per day (80 μg/kg · d), or 20 μg/kg of hPTH(1-34) per day (20 μg/kg · d). With the regimens of 80 μg/kg per dose and 80 μg/kg · d, high-frequency hPTH(1-34) administration increased metaphyseal trabecular number. However, 4 doses per day induced the formation of thin trabeculae, whereas the daily PTH regimen resulted in thicker trabeculae. A similar pattern was observed with the lower daily hPTH(1-34) dose (20 μg/kg · d): more frequent PTH administration led to the formation of thin trabeculae, showing a thick preosteoblastic cell layer, several osteoclasts, and scalloped cement lines that indicated accelerated bone remodeling. On the other hand, low-frequency PTH administration induced new bone with mature osteoblasts lying on mildly convex surfaces representative of arrest lines, which suggests minimodeling-based bone formation. Thus, high-frequency PTH administration seems to increase bone mass rapidly by forming thin trabeculae through accelerated bone remodeling. Alternatively, low-frequency PTH administration leads to the formation of thicker trabeculae through bone remodeling and minimodeling.

Formation of an alternate lamellar pattern in the advanced cellular cementogenesis in human teeth.

Abstract The formation of an alternate lamellar pattern in the advanced stage of cellular cementogenesis in human molars was examined by light and electron microscopy. In longitudinal ultrathin sections, longitudinally oriented intrinsic fibril bundles appeared in close and parallel association with slender processes of cementoblasts on the cementum. Where transversely oriented intrinsic fibril bundles appeared, cementoblasts formed indentations to enclose the fibril bundles. Cytoplasmic fragments were also enclosed in the indentations. Scanning electron microscopy indicated that cementoblasts have developed two types of processes on their cementum-facing side – ridge- and finger-like. The cementoblasts formed groove-like compartments by ridge-like processes in cooperation with other cementoblasts. The compartments formed groups, and in each group the compartments were arranged in the same direction. The finger-like processes were arranged in parallel with the ridge-like processes in the compartments. These observations suggest that: (1) slender processes and cytoplasmic fragments are longitudinally and transversely cut finger-like processes, respectively; (2) the cellular indentations are transversely cut groove-like compartments; (3) the cementoblasts regulate the intrinsic fiber arrangement by the two types of processes; (4) the cementoblasts move the two types of processes synchronously and periodically to cause an alternate change in the intrinsic fiber arrangement. This dynamic sequence results in the alternate lamellar pattern of cellular cementum.

Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization

Summary Matrix vesicle-mediated mineralization is an orchestrated sequence of ultrastructural and biochemical events that lead to crystal nucleation and growth. The influx of phosphate ions into the matrix vesicle is mediated by several proteins such as TNAP, ENPP1, Pit1, annexin and so forth. The catalytic activity of ENPP1 generates pyrophosphate (PPi) using extracellular ATPs as a substrate, and the resultant PPi prevents crystal overgrowth. However, TNAP hydrolyzes PPi into phosphate ion monomers, which are then transported into the matrix vesicle through Pit1. Accumulation of Ca2+ and PO43− inside matrix vesicles then induces crystalline nucleation, with calcium phosphate crystals budding off radially, puncturing the matrix vesicle’s membrane and finally growing out of it to form mineralized nodules.

Localization of Minodronate in Mouse Femora Through Isotope Microscopy

Minodronate is highlighted for its marked and sustained effects on osteoporotic bones. To determine the duration of minodronate’s effects, we have assessed the localization of the drug in mouse bones through isotope microscopy, after labeling it with a stable nitrogen isotope ([15N]-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and transmission electron microscopy (TEM). Eight-week-old male ICR mice received [15N]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [15N]-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hr after minodronate administration, histochemistry and TEM showed osteoclasts with well-developed ruffled borders. However, osteoclasts were roughly attached to the bone surfaces and did not feature ruffled borders at 24 hr after minodronate administration. The numbers of tartrate-resistant acid phosphatase–positive osteoclasts and alkaline phosphatase–reactive osteoblastic area were not reduced suddenly, and apoptotic osteoclasts appeared in 1 week and 1 month after the injections. Von Kossa staining demonstrated that osteoclasts treated with minodronate did not incorporate mineralized bone matrix. Taken together, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect.

Histology of human cementum: Its structure, function, and development

Summary Cementum was first demonstrated by microscopy, about 180 years ago. Since then the biology of cementum has been investigated by the most advanced techniques and equipment at that time in various fields of dental sciences. A great deal of data on cementum histology have been accumulated. These data have been obtained from not only human, but also non-human animals, in particular, rodents such as the mouse and rat. Although many dental histologists have reviewed histology of human cementum, some descriptions are questionable, probably due to incorrect comparison of human and rodent cementum. This review was designed to introduce current histology of human cementum, i.e. its structure, function, and development and to re-examine the most questionable and controversial conclusions made in previous reports.

Hertwig's Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.

Hertwig’s epithelial root sheath cell behavior during initial acellular cementogenesis in rat molars

This study was designed to examine developing acellular cementum in rat molars by immunohistochemistry, to elucidate (1) how Hertwig’s epithelial root sheath disintegrates and (2) whether epithelial sheath cells transform into cementoblasts through epithelial–mesenchymal transition (EMT). Initial acellular cementogenesis was divided into three developmental stages, which can be seen in three different portions of the root: portion 1, where the epithelial sheath is intact; portion 2, where the epithelial sheath becomes fragmented; and portion 3, where acellular cementogenesis begins. Antibodies against three kinds of matrix proteinases, which degrade epithelial sheath-maintaining factors, including basement membrane and desmosomes, were used to investigate proteolytic activity of the epithelial sheath. Tissue non-specific alkaline phosphatase (TNALP) and keratin were used to investigate EMT. Epithelial sheath cells showed immunoreactivity for all three enzymes at fragmentation, which suggests that epithelial sheath disintegration is enzymatically mediated. Dental follicle cells and cementoblasts showed intense immunoreactivity for TNALP, and from portion 1 through to 3, the reaction extended from the alveolar bone-related zone to the root-related zone. Cells possessing keratin/TNALP double immunoreactivity were virtually absent. Keratin-positive epithelial sheath cells showed negligible immunoreactivity for TNALP, and epithelial cells did not appear to migrate to the dental follicle. Together, these findings suggest that a transition phenotype between epithelial cells and cementoblasts does not exist in the developing dental follicle and hence that epithelial sheath cells do not undergo EMT during initial acellular cementogenesis. In brief, this study supports the notion that cementoblasts derive from the dental follicle.

Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM)

The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes’ cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.