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.

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.

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.

Histochemical assessment on the cellular interplay of vascular endothelial cells and septoclasts during endochondral ossification in mice.

This study was aimed to verify the cellular interplay between vascular endothelial cells and surrounding cells in the chondro-osseous junction of murine tibiae. Many CD31-positive endothelial cells accompanied with Dolichos Biflorus Agglutinin lectin-positive septoclasts invaded into the hypertrophic zone of the tibial epiphyseal cartilage. MMP9 immunoreactive cytoplasmic processes of vascular endothelial cells extended into the transverse partitions of cartilage columns. In contrast, septoclasts included several large lysosomes which indicate the incorporation of extracellular matrices despite no immunopositivity for F4/80-a hallmark of macrophage/monocyte lineage. In addition, septoclasts were observed in c-fos-/- mice but not in Rankl-/- mice. Unlike c-fos-/- mice, Rankl-/- mice showed markedly expanded hypertrophic zone and the irregular shape of the chondro-osseous junction. Immunoreactivity of platelet-derived growth factor-bb, which involved in angiogenic roles in the bone, was detected in not only osteoclasts but also septoclasts at the chondro-osseous junction. Therefore, septoclasts appear to assist the synchronous vascular invasion of endothelial cells at the chondro-osseous junction. Vascular endothelial cells adjacent to the chondro-osseous junction possess endomucin but not EphB4, whereas those slightly distant from the chondro-osseous junction were intensely positive for both endomucin and EphB4, while being accompanied with ephrinB2-positive osteoblasts. Taken together, it is likely that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2. MINI-ABSTRACT: Our original article demonstrated that vascular endothelial cells adjacent to the chondro-osseous junction would interplay with septoclasts for synchronous invasion into the epiphyseal cartilage, while those slightly distant from the chondro-osseous junction would cooperate with osteoblastic activities presumably by mediating EphB4/ephrinB2. (A figure that best represents your paper is Fig. 5c).

Immunocytochemical assessment of cell differentiation of podoplanin-positive osteoblasts into osteocytes in murine bone.

In this study, we examined the immunolocalization of podoplanin/E11, CD44, actin filaments, and phosphorylated ezrin in the osteoblasts on the verge of differentiating into osteocytes in murine femora and tibiae. When observing under stimulated emission depletion microscopy, unlike podoplanin-negative osteoblasts, podoplanin-positive osteoblasts showed a rearranged assembly of actin filaments along the cell membranes which resembled that of embedded osteocytes. In the metaphysis, i.e., the bone remodeling site, CD44-bearing osteoclasts were either proximal to or in contact with podoplanin-positive osteoblasts, but the podoplanin-positive osteoblasts also localized CD44 on their own cell surface. These podoplanin-positive osteoblasts, which either possessed CD44 on their cell surface or were close to CD44-bearing osteoclasts, showed phosphorylated ezrin-positivity on the cell membranes. Therefore, the CD44/podoplanin interaction on the cell surface may be involved in the osteoblastic differentiation into osteocytes in the metaphyses, via the mediation of podoplanin-driven ezrin phosphorylation and the subsequent reorganized assembly of actin filaments. Consistently, the protein expression of phosphorylated ezrin was increased after CD44 administration in calvarial culture. Conversely, in modeling sites such as the cortical bones, podoplanin-positive osteoblasts were uniformly localized at certain intervals even without contact with CD44-positive bone marrow cells; furthermore, they also exhibited phosphorylated ezrin immunoreactivity along their cell membranes. Taken together, it seems likely that the CD44/podoplanin interaction is involved in osteoblastic differentiation into osteocytes in the bone remodeling area but not in modeling sites.

Histochemical assessment for osteoblastic activity coupled with dysfunctional osteoclasts in c-src deficient mice.

Since osteoblastic activities are believed to be coupled with osteoclasts, we have attempted to histologically verify which of the distinct cellular circumstances, the presence of osteoclasts themselves or bone resorption by osteoclasts, is essential for coupled osteoblastic activity, by examining c-fos-/- or c-src-/- mice. Osteopetrotic c-fos deficient (c-fos-/-) mice have no osteoclasts, while c-src deficient (c-src-/-) mice, another osteopetrotic model, develop dysfunctional osteoclasts due to a lack of ruffled borders. c-fos-/- mice possessed no tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts, and showed very weak tissue nonspecific alkaline phosphatase (TNALPase)-reactive mature osteoblasts. In contrast, c-src-/- mice had many TNALPase-positive osteoblasts and TRAPase-reactive osteoclasts. Interestingly, the parallel layers of TRAPase-reactive/osteopontin-positive cement lines were observed in the superficial region of c-src-/- bone matrix. This indicates the possibility that in c-src-/- mice, osteoblasts were activated to deposit new bone matrices on the surfaces that osteoclasts previously passed along, even without bone resorption. Transmission electron microscopy demonstrated cell-to-cell contacts between mature osteoblasts and neighboring ruffled border-less osteoclasts, and osteoid including many mineralized nodules in c-src-/- mice. Thus, it seems likely that osteoblastic activities would be maintained in the presence of osteoclasts, even if they are dysfunctional.

Histological assessment for femora of ovariectomized obesity (db/db) mice carrying mutated leptin receptor.

In order to provide a clue to understand the interplay between leptin and estrogen, we have examined femoral metaphyses of ovariectomized db/db mice carrying a mutated leptin receptor. We performed ovariectomy (OVX) or sham-operation (sham) on 12-week old female wild-type and db/db mice, and then, after 8 weeks, divided the animals into four groups: wild-type sham, wild-type OVX, db/db sham and db/db OVX. Samples from all groups were prepared for histochemical and ultrastructural examinations. As a result, db/db sham mice showed a reduced number and thickness of metaphyseal trabeculae and excessive adipose tissue when compared to wild-type sham mice. The wild-type OVX group exhibited markedly diminished trabecular number, as well as lower populations of osteoblasts and osteoclasts in comparison to wild-type sham group. On the other hand, trabecular numbers were similar for the two db/db groups, suggesting that the effect of the ovariectomy, i.e., estrogen deficiency may be lessened in this animal model. Leptin receptor was mainly found in osteoblasts and in bone marrow stromal cells including adipocytes. In addition, the expression of estrogen receptor did not seem to change after OVX in wild-type mice and in db/db mice. Both db/db sham and OVX mice featured many adipocytes close to the metaphyseal chondro-osseous junction, while osteoblasts accumulated glycogen granules and lipid droplets. Therefore, it seems likely that the disruption of leptin signaling in db/db mice shifts the cell differentiation cascade towards the adipocyte lineage, resulting in an osteoporotic bone independently of estrogen deficiency.

Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats.

Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous α-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodules, and were apparently bound to collagenous fibrillar structures. In summary, collagen mineralization seems unaffected by ascorbic acid deficiency in spite of the fine, fragile collagenous fibrils identified in the bones of our animal model.