Search

Objectives: This study aimed to elucidate whether the administration of parathyroid hormone (PTH) results in remodeling- or modeling-based bone formation in different regions of the murine femora, and whether the PTH-driven bone formation would facilitate osteoblastic differentiation into osteocytes., Methods: Six-week-old male C57BL/6J mice were employed to examine the distribution of alkaline phosphatase (ALP), PHOSPHO1, podoplanin, and calcein labeling in two distinct long bone regions: the metaphyseal trabeculae close to the chondro-osseous junction (COJ) and those distant from the COJ in three mouse groups, a control group receiving a vehicle (sham group) and groups receiving hPTH (1-34) twice a day (PTH BID group) or four times a day (PTH QID group) for two weeks., Results: The sham group showed PHOSPHO1-reactive mature osteoblasts localized primarily at the COJ, whereas the PTH BID/QID groups exhibited extended lines of PHOSPHO1-reactive osteoblasts even in regions distant from the COJ. The PTH QID group displayed fragmented calcein labeling in trabeculae close to the COJ, whereas continuous labeling was observed in trabeculae distant from the COJ. Osteoblasts tended to express podoplanin and PHOSPHO1 independently in the close and distant regions of the sham group, while osteoblasts in the PTH-administered groups showed immunoreactivity of podoplanin and PHOSPHO1 together in the close and distant regions., Conclusions: Administration of PTH may accelerate remodeling-based bone formation in regions close to the COJ while predominantly inducing modeling-based bone formation in distant regions. PTH appeared to simultaneously facilitate osteoblastic bone mineralization and differentiation into osteocytes in both remodeling- and modeling-based bone formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest None of the authors declares a conflict of interest., (Copyright © 2023 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest No potential conflicts of interest exist., (Copyright © 2023 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflicts of interest The authors have no conflict of interest., (Copyright © 2023 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2023 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflicts of interest No potential conflicts of interest exist., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier B.V.)

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., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2021 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)