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.

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.

Bone biopsy findings in patients receiving long-term bisphosphonate therapy for glucocorticoid-induced osteoporosis.

INTRODUCTION: Bisphosphonates (BPs) have been shown to reduce the incidence of vertebral fractures during the first year or two of glucocorticoid (GC) treatments and are therefore recommended as a first-line treatment for GC-induced osteoporosis (GIO). However, there are theoretical concerns about the long-term use of BPs in low-turnover osteoporosis caused by chronic GC therapy. MATERIALS AND METHODS: We analyzed the trabecular microarchitecture, bone metabolism, and material strength of iliac crest bone biopsy samples from 10 female patients with rheumatoid arthritis who received an average of 6.7 years of BP therapy for GIO (GIOBP group), compared with those of 10 age- and bone mineral density (BMD)-matched non-rheumatoid arthritis postmenopausal women (reference group). RESULTS: Patients in the GIOBP group had a significantly greater fracture severity index, as calculated from the number and the extent of vertebral fractures compared with the reference patients. Micro-computed tomography analysis showed that the degree of mineralization and trabecular microarchitecture were significantly lower in the GIOBP group than in the reference patients. Patients in the GIOBP group exhibited lower bone contact stiffness, determined by micro-indentation testing, than in the reference group. The contact stiffness of the bone was negatively correlated with the fracture severity index and the daily prednisolone dosage. Immunohistochemistry and serum bone turnover markers showed decreased osteoclastic activity, impaired mineralization, and an increased fraction of empty lacunae in the GIOBP group. CONCLUSION: Our findings indicate that patients receiving long-term BP for GIO are still at high risk for fragility fractures because of poor bone quality.

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.

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.

Intermittent PTH Administration Increases Bone-Specific Blood Vessels and Surrounding Stromal Cells in Murine Long Bones.

To verify whether PTH acts on bone-specific blood vessels and on cells surrounding these blood vessels, 6-week-old male mice were subjected to vehicle (control group) or hPTH [1-34] (20 µg/kg/day, PTH group) injections for 2 weeks. Femoral metaphyses were used for histochemical and immunohistochemical studies. In control metaphyses, endomucin-positive blood vessels were abundant, but αSMA-reactive blood vessels were scarce. In the PTH-administered mice, the lumen of endomucin-positive blood vessels was markedly enlarged. Moreover, many αSMA-positive cells were evident near the blood vessels, and seemed to derive from those vessels. These αSMA-positive cells neighboring the blood vessels showed features of mesenchymal stromal cells, such as immunopositivity for c-kit and tissue nonspecific alkaline phosphatase (TNALP). Thus, PTH administration increased the population of perivascular/stromal cells positive for αSMA and c-kit, which were likely committed to the osteoblastic lineage. To understand the cellular events that led to increased numbers and size of bone-specific blood vessels, we performed immunohistochemical studies for PTH/PTHrP receptor and VEGF. After PTH administration, PTH/PTHrP receptor, VEGF and its receptor flk-1 were consistently identified in both osteoblasts and blood vessels (endothelial cells and surrounding perivascular cells). Our findings suggest that exogenous PTH increases the number and size of bone-specific blood vessels while fostering perivascular/stromal cells positive for αSMA/TNALP/c-kit.

Altered immunolocalization of FGF23 in murine femora metastasized with human breast carcinoma MDA-MB-231 cells.

INTRODUCTION: After the onset of bone metastasis, tumor cells appear to modify surrounding microenvironments for their benefit, and particularly, the levels of circulating fibroblast growth factor (FGF) 23 in patients with tumors have been highlighted. MATERIALS AND METHODS: We have attempted to verify if human breast carcinoma MDA-MB-231 cells metastasized in the long bone of nu/nu mice would synthesize FGF23. Serum concentrations of calcium, phosphate (Pi) and FGF23 were measured in control nu/nu mice, bone-metastasized mice, and mice with mammary gland injected with MDA-MB-231 cells mimicking primary mammary tumors. RESULTS AND CONCLUSIONS: MDA-MB-231 cells revealed intense FGF23 reactivity in metastasized lesions, whereas MDA-MB-231 cells cultured in vitro or when injected into the mammary glands (without bone metastasis) showed weak FGF23 immunoreactivity. Although the bone-metastasized MDA-MB-231 cells abundantly synthesized FGF23, osteocytes adjacent to the FGF23-immunopositive tumors, unlike intact osteocytes, showed no FGF23. Despite significantly elevated serum FGF23 levels in bone-metastasized mice, there was no significant decrease in the serum Pi concentration when compared with the intact mice and mice with a mass of MDA-MB-231 cells in mammary glands. The metastasized femora showed increased expression and FGFR1 immunoreactivity in fibroblastic stromal cells, whereas femora of control mice showed no obvious FGFR1 immunoreactivity. Taken together, it seems likely that MDA-MB-231 cells synthesize FGF23 when metastasized to a bone, and thus affect FGFR1-positive stromal cells in the metastasized tumor nest in a paracrine manner.

Sequential Treatment with Eldecalcitol After PTH Improves Bone Mechanical Properties of Lumbar Spine and Femur in Aged Ovariectomized Rats.

Parathyroid hormone (PTH) analogs have a powerful anabolic effect on bone and are used in the treatment of patients with severe osteoporosis. However, there are limitations to how long they can be safely administered. Withdrawal of PTH results in the cancelation of its effects, necessitating subsequent treatment to maintain the bone quantity and quality. This study assessed the effects of Eldecalcitol (ELD), an active vitamin D3 derivative, after PTH in estrogen-deficient osteoporotic rats. Six-month-old female rats were ovariectomized, and PTH administration was started 7 weeks later. After 4 weeks of PTH treatment, the animals were divided into three groups and either continued to receive PTH (PTH-PTH), or were switched to ELD (PTH-ELD) or vehicle (PTH-Veh) for an additional 4 weeks. In the femur, increased BMD by 4 weeks treatment of PTH was significantly reduced in PTH-Veh but not in PTH-PTH and PTH-ELD. The same tendency was observed in the lumbar vertebrae. MicroCT imaging and histomorphometry analysis revealed that the favorable bone structure changes by PTH administration were also maintained in the femurs and tibias of the PTH-PTH and PTH-ELD groups. Increased bone strength by 4-week treatment of PTH in lumber also maintained in PTH-ELD. Furthermore, minimodeling was observed in the PTH-ELD group. These results demonstrate that treatment with ELD sequentially following PTH prevented the bone quantity and strength reduction that accompanies PTH withdrawal in estrogen-deficient rats.

Histochemical examination on the peri-implant bone with early occlusal loading after the immediate placement into extraction sockets.

Early and immediate loading of dental implants has become a routine procedure in dental practices throughout the world, but the histological feature of peri-implant bone has not been fully understood. Therefore, we aimed to elucidate the histological response of peri-implant bone bearing the early occlusal loading using rat models. Four-week-old male Wistar rats were subjected to extraction of their maxillary left first molars and had titanium implants inserted immediately into the post-extraction sockets. In experimental groups at 1 week after placement, implants were loaded for 1 or 2 weeks by adding adhesive resin on the top of the screws. In control groups, no adhesive resin was added to the implants. After 1 or 2 weeks with loading, rats were fixed with an aldehyde solution for histochemical assessment. Newly-formed bone adhered broadly to the implant surface in both the control and experimental groups. The experimental group loaded for 2 weeks showed thicker trabeculae between the implant threads compared to those in the control group. Osteopontin- and osteocalcin-positive cement lines, which are histological hallmarks of bone remodeling, were narrow and smooth in the experimental groups, while featuring a complex meshwork with thick scalloped lines in the control groups. The index of sclerostin-positive osteocytes located close to implants loaded for 2 weeks was significantly lower than in controls, suggesting that osteoblast activity was preserved. Summarizing, our experimental model suggested that early implant loading increases trabecular thickness in the peri-implant bone tissue in a process that involves the regulation of bone remodeling.

Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM).

The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes' cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes.

Effects of drug discontinuation after short-term daily alendronate administration on osteoblasts and osteocytes in mice.

In order to determine whether osteoclastic bone resorption is restarted after withdrawn of bisphosphonates, we conducted histological examinations on murine osteoclasts, osteoblasts and osteocytes after discontinuation of a daily regimen of alendronate (ALN) with a dosage of 1 mg/kg/day for 10 days. After drug discontinuation, metaphyseal trabecular number and bone volume remained unaltered for the first 4 days. Osteoclast number did not increase, while the number of apoptotic osteoclasts was elevated. On the other hand, tissue non-specific alkaline phosphatase-immunoreactive area was markedly reduced after ALN discontinuation. In addition, osteocytes showed an atrophic profile with empty lacunar areas during and after ALN treatment. Interestingly, as early as 36 h after a single ALN injection, osteocytes show signs of atrophy despite the presence of active osteoblasts. Structured illumination microscopy system showed shortening of osteocytic cytoplasmic processes after drug cessation, suggesting a possible morphological and functional disconnection between osteocytes and osteoblasts. Taken together, it appears that osteoclastic bone resorption is not resumed after ALN discontinuation; also, osteoblasts and osteocytes hardly seem to recover once they are inactivated and atrophied by ALN. In summary, it seems that one must pay more attention to the responses of osteoblasts and osteocytes, rather focusing on the resuming of osteoclastic bone resorption after the ALN discontinuation.

[Vascular Calcification - Pathological Mechanism and Clinical Application - . Vascular calcification in klotho deficient environment].

Klotho deficient (kl/kl) mice exhibit Möncheberg's vascular calcification in the tunica media due to hyperphosphatemia and hypercalcemia by mediating the disrupted signaling of FGF23/klotho axis. Recent studies have hypothesized the mechanism of medial vascular calcification : Vascular smooth muscle cells acquired excessive intake of phosphate ions undergo a phenotypic differentiation into osteoblasts and induce biological calcification in the tunica media. It is useful to clarify the underlying cellular mechanism of vascular calcification for the development of the treatment and preventive medicine. This review will introduce the histological and ultrastructual findings on medial vascular calcification in kl/kl mice.

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.

[Bone and Stem Cells. Cellular network in bone micro-environment - histological and ultrastructural aspects -].

Bone micro-environment appears to reflect bone turnover, i.e., frequency of bone remodeling. There are many bone-synthesizing mature osteoblasts, bone-resorbing osteoclasts, and a thick cell layer of preosteoblasts overlying mature osteoblasts in the region which shows active bone remodeling. Bone lining cells, - flattened, resting form of osteoblasts cover the quiescent bone surface, in which, however, osteocyte-lacunar canalicular system tend to be geometrically well-arranged. Thus, bone micro-environment seems to be regulated by preosteoblasts, bone marrow stromal cells and vascular endothelial cells, as well as osteoblasts and osteoclasts. But, precious biological function of preosteoblasts and bone marrow stromal cells are still under the investigation, e.g., due to many phenotypes of preosteoblasts. In this review, we will introduce histological and ultrastructural aspects on cellular involvement in bone micro-environment.

[Microscopic aspects on biomineralization in bone].

In bone, biomineralization induced by osteoblasts is known to be initiated by small extracellular vesicles referred to as "matrix vesicles". Matrix vesicles possess many enzymes and transporters, which synthesize and incorporate Ca²âº and PO4⁻ into the vesicles. Calcification initiates when crystalline calcium phosphates are nucleated inside these matrix vesicles, and calcium phosphates, i.e., hydroxyapatite crystals, grow and eventually break through the membrane to get out of the matrix vesicles. Exposed calcium phosphates featuring "ribbon-like" appearance assemble radially, forming spherical mineralized structure, referred to as "mineralized nodule" or "calcifying globule". This process is called "matrix vesicle mineralization". Thereafter, the mineralized nodules make contacts with surrounding collagen fibrils, extending mineralization along with their longitudinal axis from the contact points of collagen fibrils - collagen mineralization. Matrix vesicle mineralization and subsequent collagen mineralization are classified as primary mineralization associated with osteoblastic bone formation. After primary mineralization, secondary mineralization takes place, gradually increasing mineral density of bone matrix. This review will introduce the microscopic findings on matrix vesicle mineralization and subsequent collagen mineralization.

Abaloparatide promotes bone repair of vertebral defects in ovariectomized rats by increasing bone formation.

Osteoporotic vertebral fracture (OVF) is the most common type of osteoporotic fracture and is associated with immobility and mortality. Bone anabolic agents, such as abaloparatide (ABL), are usually administered to patients with OVF to prevent subsequent fractures. Although several studies have shown that bone anabolic agents promote healing of long bone fractures, there is little evidence of their healing effect on vertebral bone fractures. In the present study, we investigated the effect of ABL on vertebral bone defects using ovariectomized (OVX) rats with vertebral body drill-hole defects, an animal model of OVF. Eight-week-old female Sprague-Dawley rats were subjected to OVX, followed by the 32-36 days of bone depletion period, once-daily subcutaneous ABL was administered to OVX rats at a dose of 30 µg/kg for a maximum of 6 weeks from the day of the vertebral defect surgery. We found that ABL significantly increased bone mineral content and improved trabecular structural parameters at the vertebral defect site. Moreover, ABL significantly increased bone strength of the defected vertebrae. Bone histochemical analysis revealed formation of thick trabecular bone networks at the defect site after ABL administration, consistent with an improvement in trabecular structural parameters by ABL. ABL increased ALPase- and PHOSPHO1-positive osteoblastic cells and ALPase/PCNA double-positive cells, indicating enhanced preosteoblast proliferation as well as bone formation at the defect site. On the other hand, ABL did not affect the number of cathepsin K-positive osteoclasts per bone surface, suggesting that ABL did not promote excessive bone resorption. Our findings suggest that ABL is useful not only for preventing secondary vertebral fractures but also for promoting bone healing in OVF.

Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor.

DNAX-associated protein 12 kD size (DAP12) is a dominant immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis. Although several DAP12-associated receptors (DARs) have been identified in osteoclasts, including triggering receptor expressed on myeloid cells 2 (TREM-2), C-type lectin member 5 A (CLEC5A), and sialic acid-binding Ig-like lectin (Siglec)-15, their precise role in the development of osteoclasts and bone remodeling remain poorly understood. In this study, mice deficient in Trem-2, Clec5a, Siglec-15 were generated. In addition, mice double deficient in these DAR genes and FcεRI gamma chain (FcR)γ, an alternative ITAM adaptor to DAP12, were generated. Bone mass analysis was conducted on all mice. Notably, Siglec-15 deficient mice and Siglec-15/FcRγ double deficient mice exhibited mild and severe osteopetrosis respectively. In contrast, other DAR deficient mice showed normal bone phenotype. Likewise, osteoclasts from Siglec-15 deficient mice failed to form an actin ring, suggesting that Siglec-15 promotes bone resorption principally by modulating the cytoskeletal organization of osteoclasts. Furthermore, biochemical analysis revealed that Sigelc-15 activates macrophage colony-stimulating factor (M-CSF)-induced Ras-associated protein-1 (RAP1)/Ras-related C3 botulinum toxin substrate 1 (Rac1) pathway through formation of a complex with p130CAS and CrkII, leading to cytoskeletal remodeling of osteoclasts. Our data provide genetic and biochemical evidence that Siglec-15 facilitates M-CSF-induced cytoskeletal remodeling of the osteoclasts.

Regional difference in the distribution of alkaline phosphatase, PHOSPHO1, and calcein labeling in the femoral metaphyseal trabeculae in parathyroid hormone-administered mice.

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.

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.

[Bone histology after intermittent PTH treatment - animal models -].

Intermittent administration of parathyroid hormone, PTH, appears to promote preosteoblastic proliferation and osteoblastic bone formation, giving rise to anabolic effect in bone. We investigated the behavior of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. As a consequence, bone formation was increased in PTH-treated wild-type mice, whereas in the osteoclast-deficient c-fos - / - mice, there was no significant increase in bone formation, despite the highly-increased population of preosteoblasts. It seems likely that the absence of osteoclasts might hinder PTH-driven bone anabolism, and also that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration. We will also discuss the pivotal role of osteocytes in PTH-mediated anabolic effect.