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.

Osteocalcin is necessary for the alignment of apatite crystallites, but not glucose metabolism, testosterone synthesis, or muscle mass.

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.

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.

Deletion of Tfam in Prx1-Cre expressing limb mesenchyme results in spontaneous bone fractures.

INTRODUCTION: Osteoblasts require substantial amounts of energy to synthesize the bone matrix and coordinate skeleton mineralization. This study analyzed the effects of mitochondrial dysfunction on bone formation, nano-organization of collagen and apatite, and the resultant mechanical function in mouse limbs. MATERIALS AND METHODS: Limb mesenchyme-specific Tfam knockout (Tfamf/f;Prx1-Cre: Tfam-cKO) mice were analyzed morphologically and histologically, and gene expressions in the limb bones were assessed by in situ hybridization, qPCR, and RNA sequencing (RNA-seq). Moreover, we analyzed the mitochondrial function of osteoblasts in Tfam-cKO mice using mitochondrial membrane potential assay and transmission electron microscopy (TEM). We investigated the pathogenesis of spontaneous bone fractures using immunohistochemical analysis, TEM, birefringence analyzer, microbeam X-ray diffractometer and nanoindentation. RESULTS: Forelimbs in Tfam-cKO mice were significantly shortened from birth, and spontaneous fractures occurred after birth, resulting in severe limb deformities. Histological and RNA-seq analyses showed that bone hypoplasia with a decrease in matrix mineralization was apparent, and the expression of type I collagen and osteocalcin was decreased in osteoblasts of Tfam-cKO mice, although Runx2 expression was unchanged. Decreased type I collagen deposition and mineralization in the matrix of limb bones in Tfam-cKO mice were associated with marked mitochondrial dysfunction. Tfam-cKO mice bone showed a significantly lower Young's modulus and hardness due to poor apatite orientation which is resulted from decreased osteocalcin expression. CONCLUSION: Mice with limb mesenchyme-specific Tfam deletions exhibited spontaneous limb bone fractures, resulting in severe limb deformities. Bone fragility was caused by poor apatite orientation owing to impaired osteoblast differentiation and maturation.

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.

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.

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.

Optimal administration frequency and dose of teriparatide for acceleration of biomechanical healing of long-bone fracture in a mouse model.

Despite preclinical studies demonstrating the effectiveness of teriparatide for skeletal repair in small animals, inconclusive data from clinical trials have raised questions regarding the optimal teriparatide dosing regimen for bone repair. To address this, we assessed the effect of teriparatide frequency and dose on long-bone healing using a mouse femur osteotomy/fracture model. Eight-week-old male ICR mice were subjected to open femur osteotomies, then randomized into following five groups (n = 8 per group): vehicle; low dose/high frequency: 3 µg/kg/dose, 3 times/day; low dose/low frequency: 9 µg/kg/dose, 1 time/day; high dose/high frequency: 9 µg/kg/dose, 3 times/day; high dose/low frequency: 27 µg/kg/dose, 1 time/day. Skeletal repair was assessed by microcomputed tomography, mechanical testing, and histology 4 weeks after surgery. High-dose and/or high-frequency teriparatide treatment increased callus bone volume but failed to have a significant impact on the biomechanical recovery of fractured femurs, possibly because of impaired cortical shell formation in fracture calluses. Meanwhile, low-dose/low-frequency teriparatide therapy enhanced callus bone formation without interfering with cortical shell formation despite a lesser increase in callus bone volume, leading to significant two and fourfold increases in ultimate load and stiffness, respectively. Our findings demonstrate that administering teriparatide at higher doses and/or higher frequencies raises fracture callus volume but does not always accelerate the biomechanical recovery of fractured bone, which points to the importance of finding the optimal teriparatide dosing regimen for accelerating skeletal repair.

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.

Identification of Differentially Expressed Genes Induced by Aberrant Methylation in Oral Squamous Cell Carcinomas Using Integrated Bioinformatic Analysis.

Oral squamous cell carcinoma (OSCC) is a malignant disease. Methylation plays a key role in the etiology and pathogenesis of OSCC. The goal of this study was to identify aberrantly methylated differentially expressed genes (DEGs) in OSCCs, and to explore the underlying mechanisms of tumorigenesis by using integrated bioinformatic analysis. Gene expression profiles (GSE30784 and GSE38532) were analyzed using the R software to obtain aberrantly methylated DEGs. Functional enrichment analysis of screened genes was performed using the DAVID software. Protein⁻protein interaction (PPI) networks were constructed using the STRING database. The cBioPortal software was used to exhibit the alterations of genes. Lastly, we validated the results with the Cancer Genome Atlas (TCGA) data. Twenty-eight upregulated hypomethylated genes and 24 downregulated hypermethylated genes were identified. These genes were enriched in the biological process of regulation in immune response, and were mainly involved in the PI3K-AKT and EMT pathways. Additionally, three upregulated hypomethylated oncogenes and four downregulated hypermethylated tumor suppressor genes (TSGs) were identified. In conclusion, our study indicated possible aberrantly methylated DEGs and pathways in OSCCs, which could improve the understanding of the underlying molecular mechanisms. Aberrantly methylated oncogenes and TSGs may also serve as biomarkers and therapeutic targets for the precise diagnosis and treatment of OSCCs in the future.

Characterization of a PTH1R missense mutation responsible for Jansen type metaphyseal chondrodysplasia.

Parathyroid hormone and parathyroid hormone-related peptide (PTHrP), and its receptor (PTH1R) play an important role in differentiation of bone and cartilage in the developing stages. Constitutive dimers of PTH1R are believed to be dissociated by ligand binding, and monomeric PTH1R is capable of activating G protein. Jansen type metaphyseal chondrodysplasia is caused by missense mutations in PTH1R, which are constitutively active even without the presence of the ligands. However, the underlying pathomechanisms remained largely unknown. In this study, we have attempted to further characterize a PTH1R missense mutation H223R responsible for Jansen type metaphyseal chondrodysplasia. cDNAs encoding wild-type (Wt)- and H223R mutant (Mut)-PTH1R were transfected into HEK293T cells, and as a consequence of western blots, both the Wt- and Mut-PTH1R proteins showed several fragments between 55 and 65 kDa in size, while the patterns of N-glycosylation were distinct between them. Then we hypothesized that the Mut-PTH1R might physically interact with the Wt-PTH1R, leading to affect the downstream cAMP accumulation. Co-immunoprecipitation assays clearly showed that interaction occurred not only between the Wt-PTH1R themselves, but also between the Wt- and Mut-PTH1R. Furthermore, we performed CRE reporter assays to investigate cAMP accumulation. Constitutive, ligand-independent cAMP accumulation was observed in HEK293T cells expressing the Mut-PTH1R. Interestingly, there was a statistically lower constitutive activity in HEK293T cells co-expressing the Wt- and Mut-PTH1R proteins. Summarizing, it seems likely that Mut-PTH1R may be, at least in part, co-localized with Wt-PTH1R by forming a heterodimer, leading to affect the function to each other.

[Bone remodeling and modeling/mini-modeling.]

Modeling, adapting structures to loading by changing bone size and shapes, often takes place in bone of the fetal and developmental stages, while bone remodeling-replacement of old bone into new bone-is predominant in the adult stage. Modeling can be divided into macro-modeling(macroscopic modeling)and mini-modeling(microscopic modeling). In the cellular process of mini-modeling, unlike bone remodeling, bone lining cells, i.e., resting flattened osteoblasts covering bone surfaces will become active form of osteoblasts, and then, deposit new bone onto the old bone without mediating osteoclastic bone resorption. Among the drugs for osteoporotic treatment, eldecalcitol(a vitamin D3 analog)and teriparatide(human PTH[1-34])could show mini-modeling based bone formation. Histologically, mature, active form of osteoblasts are localized on the new bone induced by mini-modeling, however, only a few cell layer of preosteoblasts are formed over the newly-formed bone, and accordingly, few osteoclasts are present in the region of mini-modeling. In this review, histological characteristics of bone remodeling and modeling including mini-modeling will be introduced.

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation.

Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition.

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.

[Cellular interplay of bone cells and vascular endothelial cells in bone].

During endochondral bone development, the longitudinal vascular invasion into cartilage primordium initially takes place, by which mineralized cartilage matrix would be exposed into bone. Thereafter, osteogenic cells differentiate into mature osteoblasts to deposit new bone onto the exposed mineralized cartilage. New bone formation at the chondro-osseous junction appears to be achieved by the process of modeling, but not by bone remodeling based on cellular coupling between osteoclasts and osteoblasts. Recently, a specific vessel subtype in bone was reported:Vascular endothelial cells close to the chondro-oseous junction showed intense CD31/Endomucin(CD31(hi)Emcn(hi), type H), while the endothelial cells of sinusoidal vessels in diaphysis revealed only weak CD31/Endomucin(CD31(lo)Emcnlo, type L). It is suggested crucial roles of endothelial HIF in controlling bone angiogenesis, type H vessel abundance, endothelial growth factor expression and osteogenesis.