Hiromi Hongo

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.

Structure and formation of the twisted plywood pattern of collagen fibrils in rat lamellar bone

This study was designed to elucidate details of the structure and formation process of the alternate lamellar pattern known to exist in lamellar bone. For this purpose, we examined basic internal lamellae in femurs of young rats by transmission and scanning electron microscopy, the latter employing two different macerations with NaOH at concentrations of 10 and 24%. Observations after the maceration with 10% NaOH showed that the regular and periodic rotation of collagen fibrils caused an alternation between two types of lamellae: one consisting of transversely and nearly transversely cut fibrils, and the other consisting of longitudinally and nearly longitudinally cut fibrils. This finding confirms the consistency of the twisted plywood model. The maceration method with 24% NaOH removed bone components other than cells, thus allowing for three-dimensional observations of osteoblast morphology. Osteoblasts extended finger-like processes paralleling the inner bone surface, and grouped in such a way that, within a group, the processes arranged in a similar direction. Transmission electron microscopy showed that newly deposited fibrils were arranged alongside these processes. For the formation of the alternating pattern, our findings suggest that: (1) osteoblasts control the collagen fibril arrangement through their finger-like process position; (2) osteoblasts behave similarly within a group; (3) osteoblasts move their processes synchronously and periodically to promote alternating different fibril orientation; and (4) this dynamic sequential deposition of fibrils results in the alternate lamellar (or twisted plywood) pattern.

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.

Morphological aspects of the biological function of the osteocytic lacunar canalicular system and of osteocyte-derived factors

Abstract Osteocytes are organized in functional syncytia collectively referred to as the osteocytic lacunar–canalicular system (OLCS). The osteocytes are interconnected through gap junctions between their cytoplasmic processes, which pass through narrow passageways referred to as osteocytic canaliculi. There are two possible ways molecules can be transported throughout the OLCS: via the cytoplasmic processes and their gap junctions, and via the pericellular space in the osteocytic canaliculi. Transport of minerals and small molecules through a spatially well-organized OLCS is vital for bone mineral homeostasis, mechanosensing, and bone remodeling control. Recently, osteocyte-derived molecules – sclerostin, dentin matrix protein-1, fibroblast growth factor 23 (FGF23) – have been put in evidence as they may be related to osteocytic functions such as mechanosensing, regulation of bone remodeling, and so forth. FGF23 regulates serum phosphate concentration by affecting renal function, while sclerostin can inhibit osteoblastic activities. In our observations, FGF23 and sclerostin synthesis seemed to be associated with the spatial regularity of the OLCS. This review will introduce our recent morphological studies on the regularity of OLCS and the synthesis of osteocyte-derived FGF23 and sclerostin.

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.

Dose effects of beta-tricalcium phosphate nanoparticles on biocompatibility and bone conductive ability of three-dimensional collagen scaffolds

Three-dimensional collagen scaffolds coated with beta-tricalcium phosphate (β-TCP) nanoparticles reportedly exhibit good bioactivity and biodegradability. Dose effects of β-TCP nanoparticles on biocompatibility and bone forming ability were then examined. Collagen scaffold was applied with 1, 5, 10, and 25 wt% β-TCP nanoparticle dispersion and designated TCP1, TCP5, TCP10, and TCP25, respectively. Compressive strength, calcium ion release and enzyme resistance of scaffolds with β-TCP nanoparticles applied increased with β-TCP dose. TCP5 showed excellent cell-ingrowth behavior in rat subcutaneous tissue. When TCP10 was applied, osteoblastic cell proliferation and rat cranial bone augmentation were greater than for any other scaffold. The bone area of TCP10 was 7.7-fold greater than that of non-treated scaffold. In contrast, TCP25 consistently exhibited adverse biological effects. These results suggest that the application dose of β-TCP nanoparticles affects the scaffold bioproperties; consequently, the bone conductive ability of TCP10 was remarkable.

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.