Immunohistochemical and Morphometric Assessment on the Biological Function and Vascular Endothelial Cells in the Initial Process of Cortical Porosity in Mice With PTH Administration.

To clarify the cellular mechanism of cortical porosity induced by intermittent parathyroid hormone (PTH) administration, we examined the femoral cortical bone of mice that received 40 µg/kg/day (four times a day) human PTH (hPTH) (1-34). The PTH-driven cortical porosity initiated from the metaphyseal region and chronologically expanded toward the diaphysis. Alkaline phosphatase (ALP)-positive osteoblasts in the control mice covered the cortical surface, and endomucin-positive blood vessels were distant from these osteoblasts. In PTH-administered mice, endomucin-reactive blood vessels with TRAP-positive penetrated the ALP-positive osteoblast layer, invading the cortical bone. Statistically, the distance between endomucin-positive blood vessels and the cortical bone surface abated after PTH administration. Transmission electron microscopic observation demonstrated that vascular endothelial cells often pass through the flattened osteoblast layer and accompanied osteoclasts in the deep region of the cortical bone. The cell layers covering mature osteoblasts thickened with PTH administration and exhibited ALP, α-smooth muscle actin (αSMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-κB ligand (RANKL). Within these cell layers, osteoclasts were found near endomucin-reactive blood vessels. In PTH-administered femora, osteocytes secreted Dkk1, a Wnt inhibitor that affects angiogenesis, and blood vessels exhibited plasmalemma vesicle-associated protein, an angiogenic molecule. In summary, endomucin-positive blood vessels, when accompanied by osteoclasts in the ALP/αSMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1.

Eldecalcitol Induces Minimodeling-Based Bone Formation and Inhibits Sclerostin Synthesis Preferentially in the Epiphyses Rather than the Metaphyses of the Long Bones in Rats.

This study aimed to examine minimodeling-based bone formation between the epiphyses and metaphyses of the long bones of eldecalcitol (ELD)-administered ovariectomized rats. Sixteen-week-old female rats were divided into four groups: sham-operated rats receiving vehicle (Sham group), ovariectomized (OVX) rats receiving vehicle (Vehicle group), or ELDs (30 or 90 ng/kg BW, respectively; ELD30 and ELD90 groups). ELD administration increased bone volume and trabecular thickness, reducing the number of osteoclasts in both the epiphyses and metaphyses of OVX rats. The Sham and Vehicle groups exhibited mainly remodeling-based bone formation in both regions. The epiphyses of the ELD groups showed a significantly higher frequency of minimodeling-based bone formation than remodeling-based bone formation. In contrast, the metaphyses exhibited significantly more minimodeling-based bone formation in the ELD90 group compared with the ELD30 group. However, there was no significant difference between minimodeling-based bone formation and remodeling-based bone formation in the ELD90 group. While the minimodeling-induced new bone contained few sclerostin-immunoreactive osteocytes, the underlying pre-existing bone harbored many. The percentage of sclerostin-positive osteocytes was significantly reduced in the minimodeling-induced bone in the epiphyses but not in the metaphyses of the ELD groups. Thus, it seems likely that ELD could induce minimodeling-based bone formation in the epiphyses rather than in the metaphyses, and that ELD-driven minimodeling may be associated with the inhibition of sclerostin synthesis.

Phosphorylated pullulan promotes calcification during bone regeneration in the bone defects of rat tibiae.

The current study aimed to evaluate bone tissue regeneration using a combination of ß-tricalcium phosphate (ßTCP) and phosphorylated pullulan (PPL, a phosphate-rich polysaccharide polymer consisting of maltotriose units). Round defects of 2 mm diameter were created in the arterial center of rat tibiae, which were further treated with vehicle (control group), ßTCP (ßTCP group), or ßTCP + PPL (ßTCP + PPL group) grafts. The control specimens without bone grafts exhibited rapid bone formation after 1 week; however, the regenerated bone was not resorbed until 4 weeks. In contrast, ßTCP-grafted specimens exhibited fewer but thicker trabeculae, whereas the ßTCP + PPL group displayed many fine trabeculae at 4 weeks. In the ßTCP + PPL group, new bone was associated with the ßTCP granules and PPL. Similarly, PHOSPHO1-positive osteoblasts were localized on the ßTCP granules as well as the PPL. On the other hand, TRAP-reactive osteoclasts predominantly localized on newly-formed bone and ßTCP granules rather than on the PPL. No significant differences were observed in the expression of Alp, Integrin αv, Osteopontin, Osteocalcin, and Dmp-1 in PPL-treated MC3T3-E1 osteoblastic cells, suggesting that PPL did not facilitate osteoblastic differentiation. However, von Kossa staining identified abundant needle-like calcified structures extending inside the PPL. Furthermore, transmission electron microscopy (TEM) revealed many globular structures identical to calcified nodules. In addition, calcified collagen fibrils were observed in the superficial layer of the PPL. Thus, PPL may serve as a scaffold for osteoblastic bone formation and promotes calcification on its surface. In conclusion, we speculated that ßTCP and PPL might promote bone regeneration and could be integrated into promising osteoconductive materials.

Early gene expression profiles of anabolic and catabolic molecules in murine bone after a single PTH injection.

The current study examined the gene expression profiles of anabolic and catabolic molecules after a single parathyroid hormone (PTH) injection in mice. No significant changes were observed in alkaline phosphatase area/tissue volume, tartrate-resistant acid phosphatase-positive osteoclasts, or static bone histomorphometry parameters. However, a sudden and significant decrease in Runx2 expression occurred at 1.5 h post-injection followed by immediate elevation, while sclerostin level was initially downregulated but gradually recovered. Meanwhile, Rankl expression initially increased and then returned to baseline. The prolonged elevation of anabolic molecules and transient increase in catabolic molecules may contribute to the anabolic effect of PTH treatment.

Differential osteoblastic activity in primary metaphyseal trabecular and secondary trabeculae of c-fos deficient mice.

OBJECTIVES: It has been highlighted that osteoblastic activities in remodeling-based bone formation are coupled with osteoclastic bone resorption while those in modeling-based bone formation are independent of osteoclasts. This study aimed to verify whether modeling-based bone formation can occur in the absence of osteoclasts. METHODS: We performed histochemical analyses on the bone of eight-week-old male wild-type and c-fos-/- mice. Histochemical analyses were conducted on primary trabeculae near the chondro-osseous junction (COJ), sites of modeling-based bone formation, and secondary trabeculae, sites of remodeling-based bone formation, in the femora and tibiae of mice. RESULTS: Alkaline phosphatase (ALP) immunoreactivity, a marker of osteoblastic lineages, was observed in the metaphyseal trabeculae of wild-type mice, while ALP was scattered throughout the femora of c-fos-/- mice. PHOSPHO1, an enzyme involved in matrix vesicle-mediated mineralization, was predominantly detected in primary trabeculae and also within short lines of osteoblasts in secondary trabeculae of wild-type mice. In contrast, femora of c-fos-/- mice showed several patches of PHOSPHO1 positivity in the primary trabeculae, but there were hardly any patches of PHOSPHO1 in secondary trabeculae. Calcein labeling was consistently observed in primary trabeculae close to the COJ in both wild-type and c-fos-/- mice; however, calcein labeling in the secondary trabeculae was only detected in wild-type mice. Transmission electron microscopic examination demonstrated abundant rough endoplasmic reticulum in the osteoblasts in secondary trabeculae of wild-type mice, but not in those of c-fos-/- mice. CONCLUSIONS: Osteoblastic activities at the sites of modeling-based bone formation may be maintained in the absence of osteoclasts.

Immunolocalization of Enzymes/Membrane Transporters Related to Bone Mineralization in the Metaphyses of the Long Bones of Parathyroid-Hormone-Administered Mice.

The present study aimed to demonstrate the immunolocalization and/or gene expressions of the enzymes and membrane transporters involved in bone mineralization after the intermittent administration of parathyroid hormone (PTH). The study especially focused on TNALP, ENPP1, and PHOSPHO1, which are involved in matrix vesicle-mediated mineralization, as well as PHEX and the SIBLING family, which regulate mineralization deep inside bone. Six-week-old male mice were subcutaneously injected with 20 µg/kg/day of human PTH (1-34) two times per day (n = 6) or four times per day (n = 6) for two weeks. Additionally, control mice (n = 6) received a vehicle. Consistently with an increase in the volume of the femoral trabeculae, the mineral appositional rate increased after PTH administration. The areas positive for PHOSPHO1, TNALP, and ENPP1 in the femoral metaphyses expanded, and the gene expressions assessed by real-time PCR were elevated in PTH-administered specimens when compared with the findings in control specimens. The immunoreactivity and/or gene expressions of PHEX and the SIBLING family (MEPE, osteopontin, and DMP1) significantly increased after PTH administration. For example, MEPE immunoreactivity was evident in some osteocytes in PTH-administered specimens but was hardly observed in control specimens. In contrast, mRNA encoding cathepsin B was significantly reduced. Therefore, the bone matrix deep inside might be further mineralized by PHEX/SIBLING family after PTH administration. In summary, it is likely that PTH accelerates mineralization to maintain a balance with elevated matrix synthesis, presumably by mediating TNALP/ENPP1 cooperation and stimulating PHEX/SIBLING family expression.

Histochemical assessment of osteoclast-like giant cells in Rankl-/- mice.

OBJECTIVES: We examined mice with gene deletion of Receptor activator of nuclear factor-κB (Rank) ligand (Rankl) to histologically clarify whether they contained progenitor cells committed to osteoclastic differentiation up to the stage requiring RANK/RANKL signaling. METHODS: The tibiae and femora of ten-week-old male wild-type, c-fos-/-, and Rankl-/- mice were used for immunohistochemistry and transmission electron microscopy (TEM). RESULTS: In Rankl-/- mice, we observed osteoclast-like giant cells, albeit in low numbers, with single or two nuclei, engulfing the mineralized extracellular matrix. TEM revealed that these giant cells contained large numbers of mitochondria, vesicles/vacuoles, and clear zone-like structures but no ruffled borders. They often engulfed fragmented bony/cartilaginous components of the extracellular matrix that had been degraded. Additionally, osteoclast-like giant cells exhibited immunoreactivity for vacuolar H+-ATPase, galectin-3, and siglec-15 but not for tartrate-resistant acid phosphatase, cathepsin K, or MMP-9, all of which are classical hallmarks of osteoclasts. Furthermore, osteoclast-like giant cells were ephrinB2-positive as they were near EphB4-positive osteoblasts that are also positive for alkaline phosphatase and Runx2 in Rankl-/- mice. Unlike Rankl-/- mice, c-fos-/- mice lacking osteoclast progenitors and mature osteoclasts had no ephrinB2-positive osteoclast-like cells or alkaline phosphatase-positive/Runx2-reactive osteoblasts. This suggests that similar to authentic osteoclasts, osteoclast-like giant cells might have the potential to activate osteoblasts in Rankl-/- mice. CONCLUSIONS: It seems plausible that osteoclast-like giant cells may have acquired some osteoclastic traits and the ability to resorb mineralized matrices even when the absence of RANK/RANKL signaling halted the osteoclastic differentiation cascade.

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice.

OBJECTIVE: Toll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2-/-) mice to gain insight into TLR2 function in biological circumstances without microbial infection. METHODS: Eight-week-old male wild-type and TLR2-/- mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis. RESULTS: TLR2-/- tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2-/- osteoclasts reduced cell fusion and bone resorption activity. CONCLUSION: It seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.

Evocalcet Rescues Secondary Hyperparathyroidism-driven Cortical Porosity in CKD Male Rats.

To elucidate the effect of evocalcet, a new oral calcimimetic to bone of secondary hyperparathyroidism (SHPT) with chronic kidney disease (CKD), the rats were 5/6 nephrectomized and fed on a high-phosphate diet. The treated rats were then divided into vehicle groups and evocalcet administered groups. The rats in the vehicle groups exhibited increased levels of serum PTH and inorganic phosphate (Pi) levels, high bone turnover, and severe cortical porosity, mimicking SHPT (CKD-SHPT rats). The cortical bone of the CKD-SHPT rats showed broad demineralization around the osteocytes, suppression of Phex/small integrin-binding ligand N-linked glycoprotein-mediated mineralization in the periphery of the osteocytic lacunae, and increased levels of osteocytic cell death, all of which were considered as the first steps of cortical porosity. In contrast, evocalcet ameliorated the increased serum PTH levels, the enlarged osteocytic lacunae, and the cortical porosity of the CKD-SHPT rats. Osteocytes of CKD-SHPT rats strongly expressed PTH receptor and Pit1/Pit2, which sense extracellular Pi, indicating that PTH and Pi affected these osteocytes. Cell death of cultured osteocytes increased in a Pi concentration-dependent manner, and PTH administration rapidly elevated Pit1 expression and enhanced osteocytic death, indicating the possibility that the highly concentrated serum PTH and Pi cause severe perilacunar osteolysis and osteocytic cell death. It is likely therefore that evocalcet not only decreases serum PTH but also reduces the exacerbation combined with PTH and Pi to the demineralization of osteocytic lacunae and osteocytic cell death, thereby protecting cortical porosity in CKD-SHPT rats.

Histochemical assessment of accelerated bone remodeling and reduced mineralization in Il-6 deficient mice.

OBJECTIVES: Interleukin-6 (IL-6) contributes to the regulation of functions in various tissues and organs. Even though IL-6 has been reported to modulate bone metabolism in previous studies, this finding is controversial. This study aims to evaluate the possible involvement of IL-6 in bone metabolism by examining the histological activity of osteoblasts and osteoclasts in the femora of Il-6 deficient (Il-6-/-) mice. METHODS: Eight-week-old male Il-6-/- mice and their wild-type littermates were fixed with a paraformaldehyde solution, and their femora were extracted for micro-CT analysis, immunohistochemistry, and real-time PCR analysis. RESULTS: Il-6-/- femora showed an increased bone volume/tissue volume (TV) but a reduced bone mineral density compared with the wild-type. Furthermore, the tissue-nonspecific alkaline phosphatase positive area/TV ratio, the expression of Runx2, Osterix, and Rankl, and the number of tartrate-resistant acid phosphatase-positive osteoclasts were all increased in the Il-6-/- mice. A considerable number of unmineralized areas within the bone matrix and abundant sclerostin-reactive osteocytes were observed in Il-6-/- femoral metaphyses but not in the wild-type. Interestingly, the gene expression of Cd206 was elevated in Il-6-/- femora, and many F4/80-positive macrophages/monocytes and CD206-immunoreactive macrophages in the primary trabeculae had migrated closer to the growth plate, where intense RANKL immunoreactivity was detected. These results suggest that, in an IL-6-deficient state, CD206-positive macrophages may differentiate into osteoclasts when in contact with RANKL-reactive osteoblastic cells. CONCLUSION: In a state of IL-6 deficiency, the population and cell activities of osteoblast, osteoclasts, and macrophages seemed to be facilitated, except for the reduced mineralization in bone.

Histochemical assessment on osteocytic osteolysis in lactating mice fed with a calcium-insufficient diet.

OBJECTIVES: This study aimed to examine if feeding lactating mice a calcium-insufficient diet while simultaneously administering alendronate (ALN) could potentially induce osteocytic osteolysis. METHODS: Lactating mice were fed calcium (Ca)-insufficient diets with or without ALN administration, and then their femurs were examined for TRAP and ALP, and observed by Kossa staining and transmission electron microscopy (TEM). Mice that had been fed a Ca-insufficient diet were then fed a 44Ca-containing diet, and their tibial sections were examined by isotope microscopy. RESULTS: Mice fed a Ca-insufficient diet had a reduced number of TRAP-positive osteoclasts after ALN administration. ALN-treated, lactating mice fed a Ca-insufficient diet had enlarged lacunae in their cortical bones, and TEM imaging demonstrated expanded regions between osteocytes and lacunar walls. In ALN-treated lactating mice fed a Ca-insufficient diet, huge areas of demineralized bone matrix occurred, centered around blood vessels in the cortical bone. Isotope microscopy showed 44Ca in the vicinity of the osteocytic lacunae, and in the broad, previously demineralized region around the blood vessels in the cortical bone of lactating mice fed a 44Ca-sufficient diet. CONCLUSIONS: Bone demineralization likely takes place in the periphery of the osteocytic lacunae and in the broad regions around the blood vessels of lactating mice when they are exposed to severely reduced serum Ca through a Ca-insufficient diet coupled with ALN administration.

Matrix Vesicle-Mediated Mineralization and Osteocytic Regulation of Bone Mineralization.

Bone mineralization entails two mineralization phases: primary and secondary mineralization. Primary mineralization is achieved when matrix vesicles are secreted by osteoblasts, and thereafter, bone mineral density gradually increases during secondary mineralization. Nearby extracellular phosphate ions (PO43-) flow into the vesicles via membrane transporters and enzymes located on the vesicles' membranes, while calcium ions (Ca2+), abundant in the tissue fluid, are also transported into the vesicles. The accumulation of Ca2+ and PO43- in the matrix vesicles induces crystal nucleation and growth. The calcium phosphate crystals grow radially within the vesicle, penetrate the vesicle's membrane, and continue to grow outside the vesicle, ultimately forming mineralized nodules. The mineralized nodules then attach to collagen fibrils, mineralizing them from the contact sites (i.e., collagen mineralization). Afterward, the bone mineral density gradually increases during the secondary mineralization process. The mechanisms of this phenomenon remain unclear, but osteocytes may play a key role; it is assumed that osteocytes enable the transport of Ca2+ and PO43- through the canaliculi of the osteocyte network, as well as regulate the mineralization of the surrounding bone matrix via the Phex/SIBLINGs axis. Thus, bone mineralization is biologically regulated by osteoblasts and osteocytes.

Histological functions of parathyroid hormone on bone formation and bone blood vessels.

BACKGROUND: The intermittent administration of parathyroid hormone (PTH) has been prescribed to osteoporotic patients due to its bone anabolic effects. In addition to its actions on bone cells, PTH appears to affect bone-specific blood vessels. These blood vessels are derived from bone marrow sinusoids, which express EphB4, a hallmark of veinous vascular endothelial cells. Given the presence of osteo-vascular interactions, it is important to elucidate the effects of PTH on bone cells and blood vessels in murine models. HIGHLIGHTS: PTH stimulates preosteoblastic proliferation and osteoblastic bone formation. The former appears to be directly affected by PTH, whereas the latter requires osteoclast-mediated coupling. The administration of PTH through high-frequency dosage schemes accelerates bone turnover featuring remodeling-based bone formation, whereas low-frequency schemes cause mainly remodeling-based and partly modeling-based bone formation. Normally, many blood vessels lack alpha smooth muscle actin (αSMA)-immunoreactive vascular muscle cells surrounding basement membranes, indicating them being capillaries. However, PTH administration increases the number of blood vessels surrounded by αSMA-positive cells. These αSMA-positive cells spread out of blood vessels and express alkaline phosphatase and c-kit, suggesting their potential to differentiate into osteogenic and vascular endothelial/perivascular cells. Unlike bone cells, αSMA-positive cells did not appear in the periphery of blood vessels in the kidney and liver, and the thickness of the tunica media did not change regardless of PTH administration. CONCLUSION: Based on the results of the study and presence of osseous-vascular interactions, PTH appears to influence not only osteoblastic cells, but also blood vessels in bone.

Morphological variety of capillary ends invading the epiphyseal plate in rat femora using scanning electron microscopy with osmium maceration.

OBJECTIVES: The function of capillary ends at the epiphyseal plate has been actively investigated. However, their morphology is still poorly understood. This study was designed to examine the capillary ends invading the epiphyseal plate three-dimensionally by scanning electron microscopy and discuss the relationship between their morphology and function. METHODS: Distal halves of the femora of eight-week-old male Wistar rats were used. The specimens were divided into two groups for transmission and scanning electron microscopy. For transmission electron microscopy, sagittal ultrathin sections were routinely prepared after the demineralization of the specimens, and the chondro-osseous junction was examined at the epiphyseal plate. For scanning electron microscopy, the specimens were sagittally freeze-cracked, osmium-macerated, and routinely processed. RESULTS: Endothelial cells of capillary ends had fine fenestrations, and hence they were distinguishable from perivascular cells (also known as septoclasts). Based on the outline and the presence or absence of pores, the capillary ends were divided into four types: closed dome, closed spire, porous dome, and porous spire. The two dome types generally occupied more than half of a lacuna, whereas the two spire types generally occupied only a small part of a lacuna. The porous types engulfed cellular remnants, indicative of degraded chondrocytes, via their pores. Some of the spire types penetrated the transverse septum. CONCLUSIONS: The morphological variety of capillary ends reflected their functional variety. Observations suggest that the capillary ends change their morphology dynamically in response to various functions, including the removal of degraded chondrocytes and perforation of transverse septa.

Immunolocalization of endomucin-reactive blood vessels and α-smooth muscle actin-positive cells in murine nasal conchae.

OBJECTIVES: Recently, the biological functions of endomucin-positive blood vessels and closely associated αSMA-positive cells in long bones have been highlighted. The surrounding tissues of the flat bones, such as nasal bones covered with mucosa and lamina propria, are different from those of the long bones, indicating the different distributions of endomucin-positive blood vessels and αSMA-reactive cells in nasal bones. This study demonstrates the immunolocalization of endomucin-reactive blood vessels and αSMA-positive cells in the nasal conchae of 3- and 7-week-old mice. METHODS: The nasal conchae of 3-week-old and 7-week-old male C57BL/6J mice were used for immunoreaction of endomucin, CD34, PDGFbb, TRAP, and c-kit. RESULTS: While we identified abundant endomucin-reactive blood vessels in the lamina propria neighboring the bone, not all were positive for endomucin. More CD34-reactive cells and small blood vessels were observed in the nasal conchae of 3-week-old mice than in those of 7-week-old mice. Some αSMA-positive cells in the nasal conchae surrounded the blood vessels, indicating vascular smooth muscle cells, while other αSMA-immunopositive fibroblastic cells were detected throughout the lamina propria. αSMA-positive cells did not co-localize with c-kit-immunoreactivity, thereby indicating that the αSMA-positive cells may be myofibroblasts rather than undifferentiated mesenchymal cells. CONCLUSIONS: Unlike long bones, nasal conchae contain endomucin-positive as well as endomucin-negative blood vessels and exhibit numerous αSMA-positive fibroblastic cells throughout the lamina propria neighboring the bone. Apparently, the distribution patterns of endomucin-positive blood vessels and αSMA-positive cells in nasal conchae are different from those in long bones.

Histochemical examination of blood vessels in murine femora with intermittent PTH administration.

OBJECTIVE: To verify the biological effects of parathyroid hormone (PTH) on the blood vessels in the bone, this study aimed to investigate histological alterations in endomucin-positive blood vessels and perivascular cells in murine femora after intermittent PTH administration. For comparison with blood vessels in the bone, we examined the distribution of endomucin-positive blood vessels and surrounding αSMA-immunoreactive perivascular cells in the liver, kidney, and aorta with or without PTH administration. METHODS: Six-week-old male C57BL/6J mice received hPTH [1-34] or vehicle for two weeks. All mice were fixed with a paraformaldehyde solution after euthanasia, and the right femora, kidney, liver, and aorta were extracted for immunohistochemical analysis of endomucin, αSMA, ephrinB2, EphB4, and HIF1α. Light microscopic observations of semi-thin sections and transmission electron microscopic (TEM) observations of ultra-thin sections were performed on the left femora. RESULTS: After intermittent PTH administration, αSMA-reactive/ephrinB2-positive stromal cells appeared around endomucin-positive/EphB4-immunoreactive blood vessels in the bone. In addition, intense immunoreactivities of EphB4 and HIF1α were seen in vascular endothelial cells after the PTH treatment. Several stromal cells surrounding PTH-treated blood vessels exhibited well-developed rough endoplasmic reticulum under TEM observations. In contrast to bone tissues, αSMA-positive stromal cells did not increase around the endomucin-positive blood vessels in the kidney, liver, or aorta, even after PTH administration. CONCLUSION: These findings show that intermittent PTH administration increases αSMA-reactive/ephrinB2-positive perivascular stromal cells in bone tissue but not in the kidney, liver, or aorta, suggesting that PTH preferentially affects blood vessels in the bone.

Histochemical characteristics on minimodeling-based bone formation induced by anabolic drugs for osteoporotic treatment.

Modeling, the changes of bone size and shape, often takes place at the developmental stages, whereas bone remodeling-replacing old bone with new bone-predominantly occurs in adults. Unlike bone remodeling, bone formation induced by modeling i.e., minimodeling (microscopic modeling in cancellous bone) is independent of osteoclastic bone resorption. Although recently-developed drugs for osteoporotic treatment could induce minimodeling-based bone formation in addition to remodeling-based bone formation, few reports have demonstrated the histological aspects of minimodeling-based bone formation. After administration of eldecalcitol or romosozumab, unlike teriparatide treatment, mature osteoblasts formed new bone by minimodeling, without developing thick preosteoblastic layers. The histological characteristics of minimodeling-based bone formation is quite different from remodeling, as it is not related to osteoclastic bone resorption, resulting in convex-shaped new bone and smooth cement lines called arrest lines. In this review, we will show histological properties of minimodeling-based bone formation by osteoporotic drugs.

Three-dimensional reconstruction of the Golgi apparatus in osteoclasts by a combination of NADPase cytochemistry and serial section scanning electron microscopy.

The three-dimensional morphology of the Golgi apparatus in osteoclasts was investigated by computer-aided reconstruction. Rat femora were treated for nicotinamide adenine dinucleotide phosphatase (NADPase) cytochemistry, and light microscopy was used to select several osteoclasts in serial semi-thin sections to investigate the Golgi apparatus by backscattered electron-mode scanning electron microscopy. Lace-like structures with strong backscattered electron signals were observed around the nuclei. These structures, observed within the Golgi apparatus, were attributed to the reaction products (i.e., lead precipitates) of NADPase cytochemistry. Features on the images corresponding to the Golgi apparatus, nuclei, and ruffled border were manually traced and three-dimensionally reconstructed using ImageJ/Fiji (an open-source image processing package). In the reconstructed model, the Golgi apparatus formed an almost-continuous structure with a basket-like configuration, which surrounded all the nuclei and also partitioned them. This peculiar three-dimensional morphology of the Golgi apparatus was discovered for the first time in this study. On the basis of the location of the cis- and trans-sides of the Golgi apparatus and the reported results of previous studies, we postulated that the nuclear membrane synthesized specific proteins in the osteoclasts and, accordingly, the Golgi apparatus accumulated around the nuclei as a receptacle.

Comparative immunolocalization of tissue nonspecific alkaline phosphatase and ectonucleotide pyrophosphatase/phosphodiesterase 1 in murine bone.

OBJECTIVE: This study aimed to demonstrate the immunolocalization and gene expression of tissue nonspecific alkaline phosphatase (TNALP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in osteoblasts, preosteoblasts, and osteocytes of murine bone to provide clues for a better understanding of the supply of phosphate ions (Pi) during bone mineralization. METHODS: Six-week-old male C57BL/6J mice (n = 6) were fixed with a paraformaldehyde solution, and the right femora were extracted for immunodetection of TNALP and ENPP1, while the left tibiae were used for reverse transcription polymerase chain reaction to evaluate Tnalp and Enpp1 gene expression. RESULTS: TNALP was intensely localized on the basolateral cell membranes of mature osteoblasts and preosteoblastic cells. There was little immunoreactivity of TNALP on the secretory surface of the osteoblasts and no TNALP reactivity in the osteocytes. In contrast, ENPP1 was observed throughout the cytoplasm of mature osteoblasts and osteocytes embedded in bone but was not observed in preosteoblasts. Together, despite the fact that the osteoid is a site of matrix vesicle-mediated mineralization, ENPP1, which inhibits mineralization by providing pyrophosphates, was localized in close proximity of the osteoid, whereas TNALP, which facilitates mineralization by providing Pi, was relatively distant from the osteoid. CONCLUSION: It seems likely that the differential localization of TNALP and ENPP1 around the osteoid observed at the microscopic level may provide preferential micro-circumstance for a balanced concentration of Pi and pyrophosphate for bone mineralization.

Histological observation on the initial stage of vascular invasion into the secondary ossification of murine femoral epiphyseal cartilage.

It remains unknown whether the histology of vascular invasion during secondary ossification of epiphyseal cartilage is the same as that seen in primary ossification; we examined the initial processes of vascular invasion of secondary ossification in the murine femora. Many endomucin-immunoreactive blood vessels gathered at the central region of the articular surface, and buds of soft tissue, including glomerular loops of endomucin-immunoreactive blood vessels and TNALPase- immunopositive osteoblastic cells accompanied by TRAP-positive osteoclasts, had begun to invade the epiphyseal cartilage. The invading soft tissues formed cartilage canals displaying MMP9 immunoreactivity in the tip region, and cartilaginous collagen fibrils were not visible in the vicinity of the vascular wall of the blood vessels. Thus, the histological profile marked by invading glomerular vasculature and the erosion of the cartilage matrix near the vascular walls during secondary ossification differs from that seen during primary ossification.