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 mu g/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, alpha-smooth muscle actin (alpha SMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-kappa B 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/alpha SMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1

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

Objectives: Recently, the biological functions of endomucin-positive blood vessels and closely associated aSMA-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 aSMA-reactive cells in nasal bones. This study demonstrates the immunolocalization of endomucin-reactive blood vessels and aSMA-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 aSMApositive cells in the nasal conchae surrounded the blood vessels, indicating vascular smooth muscle cells, while other aSMA-immunopositive fibroblastic cells were detected throughout the lamina propria. aSMA-positive cells did not co-localize with c-kit-immunoreactivity, thereby indicating that the aSMApositive cells may be myofibroblasts rather than undifferentiated mesenchymal cells. Conclusions: Unlike long bones, nasal conchae contain endomucin-positive as well as endomucinnegative blood vessels and exhibit numerous aSMA-positive fibroblastic cells throughout the lamina propria neighboring the bone. Apparently, the distribution patterns of endomucin-positive blood vessels and aSMA-positive cells in nasal conchae are different from those in long bones. (c) 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved

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

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 aSMAimmunoreactive 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, aSMA, ephrinB2, EphB4, and HIF1a. 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, aSMA-reactive/ephrinB2-positive stromal cells appeared around endomucin-positive/EphB4-immunoreactive blood vessels in the bone. In addition, intense immunoreactivities of EphB4 and HIF1a 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, aSMA-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 aSMA-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. (c) 2022 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology

Histochemical examination on principal collagen fibers in periodontal ligaments of ascorbic acid-deficient ODS-od/od rats

In this study, we aimed to clarify the role of ascorbic acid in collagen synthesis in periodontal ligaments using osteogenic disorder Shionogi (ODS)/ShiJcl-od/od rats lacking L-gulonolactone oxidase. These rats cannot synthesize ascorbic acid in vivo. Eight-week-old ODS/ShiJcl-od/od male rats were administered ascorbic acid solution at a concentration of 200 mg/dL (control group, n = 6) or ascorbic acid solution at concentration of 0.3 mg/dL (insufficient group, n = 12). Six rats of the insufficient group were then given with ascorbic acid solution at concentration of 200 mg/dL for additional 3 weeks (rescued group, n = 6), and then, their mandibles were histochemically examined. Consequently, the insufficient group specimens were seen to possess fewer collagen fibers, and silver impregnation revealed numerous fine, reticular fiber-like fibrils branching off from collagen in the periodontal ligaments. In control group, faint immunoreactivities for matrix metalloproteinase (MMP)2 and cathepsin H were seen in the periphery of blood vessels and throughout the ligament, respectively. In contrast, in the insufficient group, intense MMP2-immunoreactivity was observed to be associated with collagen fibrils in the periodontal ligaments, and cathepsin H-immunopositivity was seen in ligamentous cells. The rescued group showed abundant collagen fibers filling the periodontal ligament space. Under transmission electron microscopy, ligamentous fibroblasts incorporated collagen fibrils into tubular endosomes/lysosomes while simultaneously synthesizing collagen fibril bundles. Thus, ascorbic acid insufficiency affected the immunolocalization of cathepsin H and MMP2; however, ligamentous fibroblasts appear to possess the potential to synthesize collagen fibers when supplied with ascorbic acid

The diversity of preosteoblastic morphology : Preosteoblastic response to parathyroid hormone

The current concept of a preosteoblast is a precursor of an osteoblast, which is regarded as a transient cell type during osteoblastic differentiation. We have previously demonstrated different phenotypes of preosteoblasts expressing Runx2, ALPase, and BrdU incorporation. Transmission electron microscopy revealed following four distinct preosteoblastic cell types : 1) cells rich in rough endoplasmic reticulum (rER) but with a few vesicles and vacuoles (ERrich/vesicle-poor preosteoblasts), 2) cells extending their cytoplasmic processes connecting distant cells, with a small amount of scattered cisterns of rER and many vesicles and vacuoles (ER-poor/vesicle-rich preosteoblasts), 3) translucent cells showing few dispersed cell organelles and irregular cell shape with a translucent cytoplasm (translucent cells), and 4) small cells without developed cell organelles (small undifferentiated cells). ER-rich/vesicle-poor preosteoblasts were often closely adjacent to mature osteoblasts and therefore appeared to be ready for differentiation into osteoblasts. In contrast, after the administration of parathyroid hormone (PTH), ER-poor/vesicle-rich preosteoblasts rather than ER-rich/vesicle-poor cells significantly increased in number, forming a huge meshwork overlying mature osteoblasts. Thus, ERpoor/vesicle-rich preosteoblasts appeared to respond well to PTH. We also attempted to unveil the cellular behavior of these preosteoblasts against PTH and to dissect the role of osteoclasts on the mediation of PTH anabolic actions. PTH stimulated the proliferation of ER-poor/vesicle-rich preosteoblasts and bone formation in mature osteoblasts. However, an increased population of ER-poor/vesicle-rich preosteoblasts appears to require cell coupling from osteoclasts to differentiate into ER-rich/vesicle-poor preosteoblasts and mature osteoblasts. Without osteoclasts, PTH could induce neither preosteoblastic differentiation into mature osteoblasts nor subsequent bone formation. In this mini-review, we will introduce preosteoblasts in vivo consisting of several cell types with different ultrastructural properties and PTH action on preosteoblasts

Data from: Hypoperfusion of the adventitial vasa vasorum develops an abdominal aortic aneurysm

The aortic wall is perfused by the adventitial vasa vasorum (VV). Tissue hypoxia has previously been observed as a manifestation of enlarged abdominal aortic aneurysms (AAAs). We sought to determine whether hypoperfusion of the adventitial VV could develop AAAs. We created a novel animal model of adventitial VV hypoperfusion with a combination of a polyurethane catheter insertion and a suture ligation of the infrarenal abdominal aorta in rats. VV hypoperfusion caused tissue hypoxia and developed infrarenal AAA, which had similar morphological and pathological characteristics to human AAA. In human AAA tissue, the adventitial VV were stenotic in both small AAAs (30–49 mm in diameter) and in large AAAs (> 50 mm in diameter), with the sac tissue in these AAAs being ischemic and hypoxic. These results indicate that hypoperfusion of adventitial VV has critical effects on the development of infrarenal AAA