p53 deficiency promotes bone regeneration by functional regulation of mesenchymal stromal cells and osteoblasts.

INTRODUCTION: The detailed mechanism of the process during bone healing of drill-hole injury has been elucidated, but a crucial factor in regulating drill-hole healing has not been identified. The transcription factor p53 suppresses osteoblast differentiation through inhibition of osterix expression. In present study, we demonstrate the effects of p53 deficiency on the capacity of MSCs and osteoblasts during drill-hole healing. MATERIALS AND METHODS: Mesenchymal stromal cells (MSCs) and osteoblasts were collected from bone marrow and calvaria of p53 knockout (KO) mice, respectively. The activities of cell mobility, cell proliferation, osteoblast differentiation, and wound healing of MSCs and/or osteoblasts were determined by in vitro experiments. In addition, bone healing of drill-hole injury in KO mice was examined by micro-CT and immunohistological analysis using anti-osterix, Runx2, and sclerostin antibodies. RESULTS: KO MSCs stimulated cell mobility, cell proliferation, and osteoblast differentiation. Likewise, KO osteoblasts enhanced cell proliferation and wound healing. KO MSCs and osteoblasts showed high potency in the inflammation and callus formation phases compared to those from wild-type (WT) mice. In addition, increased expression of osterix and Runx2 was observed in KO MSCs and osteoblasts that migrated in the drill-hole. Conversely, sclerostin expression was inhibited in KO mice. Eventually, KO mice exhibited high repairability of drill-hole injury, suggesting a novel role of p53 in MSCs and osteoblasts in improving bone healing. CONCLUSION: p53 Deficiency promotes bone healing of drill-hole injury by enhancing the bone-regenerative ability of MSCs and osteoblasts.

Chemokine ligand 28 (CCL28) negatively regulates trabecular bone mass by suppressing osteoblast and osteoclast activities.

INTRODUCTION: Bone metabolism imbalances cause bone metabolism diseases, like osteoporosis, through aging. Although some chemokines are known to be involved in bone mass regulation, many have not been investigated. Thus, the present study aimed to investigate the role of chemokine ligand 28 (CCL28) on bone metabolism. MATERIALS AND METHODS: To investigate the role of CCL28 on bone metabolism, 10-week-old male wild-type and Ccl28 knockout (Ccl28 KO) mice were analyzed. Microcomputed tomography analysis and bone tissue morphometry were used to investigate the effect of Ccl28 deficiency on the bone. CCL28 localization in bone tissue was assumed by immunohistochemistry. Osteoblast and osteoclast markers were evaluated by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction. Finally, in vitro experiments using MC3T3-E1 and bone marrow macrophages revealed the direct effect of CCL28 on osteoblast and osteoclast. RESULTS: This study showed that Ccl28 deficiency significantly increased bone mass and the number of mature osteoblasts. Immunoreactivity for CCL28 was observed in osteoblasts and osteoclasts on bone tissue. Additionally, Ccl28 deficiency promoted osteoblast and osteoclast maturation. Moreover, CCL28 treatment decreased osteoblast and osteoclast activities but did not affect differentiation. CONCLUSION: In summary, this study indicated that CCL28 is one of the negative regulators of bone mass by suppressing osteoblast and osteoclast activities. These results provide important insights into bone immunology and the selection of new osteoporosis treatments.

Forced expression of mouse progerin attenuates the osteoblast differentiation interrupting ß-catenin signal pathway in vitro.

Nuclear protein, lamin A, which is a component of inner membrane on nucleoplasm, plays a role in nuclear formation and cell differentiation. The expression of mutated lamin A, termed progerin, causes a rare genetic aging disorder, Hutchinson-Gilford progeria syndrome, which shows abnormal bone formation with the decrease in a number of osteoblasts and osteocytes. However, exact molecular mechanism how progerin exerts depressive effects on osteogenesis has not been fully understood. Here, we created mouse lamin A dC50 cDNA encoding progerin that lacks 50 amino acid residues at C-terminus, transfected it in mouse preosteoblast-like MC3T3-E1 cells, and examined the changes in osteoblast phenotype. When lamin A dC50-expressed cells were cultured with differentiation-inductive medium, alkaline phosphatase (ALP) activity and mRNA levels of major osteoblast markers, type I collagen (Col1), bone sialoprotein (BSP), dentine matrix protein 1 (DMP1), and Runx2 were significantly decreased, and no mineralized nodules were detected as seen in control cells expressing empty vector. In the culture with mineralization-inductive medium, mRNA levels of BSP, osteocalcin, DMP1, Runx2, and osterix were strongly decreased parallel with loss of mineralization in lamin A dC50-expressed cells, while mineralized nodules appear at 21 days in control cells. Furthermore, lamin A dC50 expression was depressed nuclear localization of ß-catenin with the decrease of GSK-3ß phosphorylation level. These results suggest that lamin A dC50 depresses osteoblast differentiation in both early and late stages, and it negatively regulates ß-catenin activity interacting with GSK-3ß in cytoplasm.

Establishment and characterization of a C57BL/6 mouse model of bone metastasis of breast cancer.

Bone is one of the most common sites of metastasis in patients with advanced breast cancer; however, the mechanisms of bone metastasis remain to be fully elucidated. Animal models are essential research tools for investigating the mechanisms of diseases and drug actions. To date, there have only been a few reports in which C57BL/6 mice were used for the study of bone metastases of breast cancer. In the current study, we found that intracardiac inoculation of C57BL/6 mouse-derived parental E0771 breast cancer cells (E0771/Pa) frequently lead to bone metastases in C57BL/6 mice within 2 weeks. The bone-metastatic clone of E0771 (E0771/Bone) established by sequential in vivo selection demonstrated a higher bone-metastatic potential. Although there were no apparent differences in cell morphology or proliferation in monolayer cultures, E0771/Bone showed increased tumorsphere formation in suspension cultures and tumor formation in the orthotopic mammary fat pad in C57BL/6 mice compared with E0771/Pa. Furthermore, E0771/Bone expressed breast cancer stem-like cell surface markers CD24-/CD44+. These findings suggest that E0771/Bone possesses cancer stem-like properties. Quantitative PCR analysis revealed that mRNA expression of parathyroid hormone-related protein (PTHrP), the most common mediator of osteolytic bone metastases of breast cancer, was significantly upregulated in E0771/Bone. Thus, cancer stem-like properties and elevated PTHrP expression likely contribute to the enhanced metastatic potential of E0771/Bone. We believe that this new mouse model is a useful tool for in vivo studies of bone metastases of breast cancer, especially for those using genetically engineered mice with a C57BL/6 background.

Tetracyclines convert the osteoclastic-differentiation pathway of progenitor cells to produce dendritic cell-like cells.

Tetracyclines, such as doxycycline and minocycline, are used to suppress the growth of bacteria in patients with inflammatory diseases. Tetracyclines have been shown to prevent bone loss, but the mechanism involved is unknown. Osteoclasts and dendritic cells (DCs) are derived from common progenitors, such as bone marrow-derived macrophages (BMMs). In this article, we show that tetracyclines convert the differentiation pathway, resulting in DC-like cells not osteoclasts. Doxycycline and minocycline inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis of BMMs, but they had no effects on cell growth and phagocytic activity. They influenced neither the proliferation nor the differentiation of bone-forming osteoblasts. Surprisingly, doxycycline and minocycline induced the expression of DC markers, CD11c and CD86, in BMMs in the presence of RANKL. STAT5 is involved in DC differentiation induced by GM-CSF. Midostaurin, a STAT5-signaling inhibitor, and an anti-GM-CSF-neutralizing Ab suppressed the differentiation induced by GM-CSF but not by tetracyclines. In vivo, the injection of tetracyclines into RANKL-injected mice and RANKL-transgenic mice suppressed RANKL-induced osteoclastogenesis and promoted the concomitant appearance of CD11c(+) cells. These results suggested that tetracyclines prevent bone loss induced by local inflammation, including rheumatoid arthritis and periodontitis, through osteoclast-DC-like cell conversion.

LRP1-deficient leptin receptor-positive cells in periodontal ligament tissue reduce alveolar bone mass by inhibiting bone formation.

OBJECTIVE: Leptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells. DESIGN: Bone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers. RESULTS: Bone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly. CONCLUSION: LRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue.

Localization of SUMOylation factors and Osterix in odontoblast lineage cells during dentin formation and regeneration.

Small ubiquitin-related modifier (SUMO) conjugation (SUMOylation) is a post-translational modification involved in various cellular processes including the regulation of transcription factors. In this study, to analyze the involvement of SUMOylation in odontoblast differentiation, we examined the immunohistochemical localization of SUMO-1, SUMO-2/3, and Osterix during rat tooth development. At the bud and cap stages, localization of SUMOs and Osterix was hardly detected in the dental mesenchyme. At the bell stage, odontoblasts just beginning dentin matrix secretion and preodontoblasts near these odontoblasts showed intense immunoreactivity for these molecules. However, after the root-formation stage, these immunoreactivities in the odontoblasts decreased in intensity. Next, to examine whether the SUMOylation participates in dentin regeneration, we evaluated the distribution of SUMOs and Osterix in the dental pulp after cavity preparation. In the coronal pulp chamber of an untreated rat molar, odontoblasts and pulp cells showed no immunoreactivity. At 4 days after cavity preparation, positive cells for SUMOs and Osterix appeared on the surface of the dentin beneath the cavity. Odontoblast-like cells forming reparative dentin were immunopositive for SUMOs and Osterix at 1 week, whereas these immunoreactivities disappeared after 8 weeks. Additionally, we further analyzed the capacity of SUMO-1 to bind Osterix by performing an immunoprecipitation assay using C2C12 cells, and showed that Osterix could undergo SUMOylation. These results suggest that SUMOylation might regulate the transcriptional activity of Osterix in odontoblast lineage cells, and thus play important roles in odontoblast differentiation and regeneration.

Increased bone mass in mice after single injection of anti-receptor activator of nuclear factor-kappaB ligand-neutralizing antibody: evidence for bone anabolic effect of parathyroid hormone in mice with few osteoclasts.

Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo.

Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells.

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha.

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.

Administration of the bisphosphonate zoledronic acid during tooth development inhibits tooth eruption and formation and induces dental abnormalities in rats.

Bisphosphonates (BPs) are potent inhibitors of osteoclastic bone resorption and widely used for the treatment of osteoporosis and metastatic bone diseases. Recently, BPs have also been shown to benefit children with primary and secondary osteoporosis, including osteogenesis imperfecta; however, their long-term safety has not been established yet. Clinical and experimental studies have demonstrated that BPs delay or inhibit tooth eruption. The failure of tooth eruption causes several dental abnormalities. In this study, to determine the effects of BPs on tooth formation, the BP zoledronic acid (ZOL) was injected into 7- and 14-day-old rats, and the development of the mandibular teeth was examined. X-ray analysis demonstrated that ZOL inhibited the eruption of both incisors and molars and their formation, especially in the molar roots. Histological examination showed that, in ZOL-treated animals, alveolar bone remained unresorbed around tooth crowns, which injured ameloblasts and enamel matrix, leading to defects of the enamel. Furthermore, haphazard proliferation of odontogenic epithelium and mesenchyme associated with primitive tooth structures, which resembles human odontomas, was induced at the basal end of incisors but not around the molars. Tooth ankylosis to alveolar bone was occasionally observed in molars. These results suggest that administration of BPs during tooth development has the potential to inhibit tooth eruption and formation and to induce several types of dental abnormalities, which may be attributed to the altered osteoclastic activities.

An in vivo 3D micro-CT evaluation of tooth movement after the application of different force magnitudes in rat molar.

OBJECTIVE: To investigate the precise longitudinal change in the periodontal ligament (PDL) space width and three-dimensional tooth movement with continuous-force magnitudes in living rats. MATERIALS AND METHODS: Using nickel-titanium closed-coil springs for 28 days, 10-, 25-, 50-, and 100-g mesial force was applied to the maxillary left first molars. Micro-CT was taken in the same rat at 0, 1, 2, 3, 10, 14, and 28 days. The width of the PDL was measured in the pressure and tension sides from 0 to 3 days. Angular and linear measurements were used to evaluate molar position at day 0, 10, 14, and 28. The finite element model (FEM) was constructed to evaluate the initial stress distribution, molar displacement, and center of rotation of the molar. RESULTS: The initial evaluation of PDL width showed no statistical differences among different force magnitudes. Tooth movement was registered 1 hour after force application and gradually increased with time. From day 10, greater tooth movement was observed when 10 g of force was applied. The FEM showed that the center of rotation in the molar is located in the center of five roots at the apical third of the molar roots. CONCLUSION: The rat's molar movement mainly consists of mesial tipping, extrusion of distal roots, intrusion of mesial root, palatal inclination, and mesial rotation. Although the initial tooth movement after the application of different force magnitudes until day 3 was not remarkably different, 10 g of force produced more tooth movement compared with heavier forces at day 28.

Intermittent parathyroid hormone 1-34 induces oxidation and deterioration of mineral and collagen quality in newly formed mandibular bone.

Intermittent parathyroid hormone (PTH) administration is known to promote bone healing after surgical procedures. However, the mechanism and influence of PTH on the mineral and collagen quality of the jaw are not well understood. Most studies have focused on analyzing the bone density and microstructure of the mandible, and have insufficiently investigated its mineral and collagen quality. Oxidative stress activates osteoclasts, produces advanced glycation end products, and worsens mineral and collagen quality. We hypothesized that PTH induces oxidation and affects the mineral and collagen quality of newly formed mandibular bone. To test this, we examined the mineral and collagen quality of newly formed mandibular bone in rats administered PTH, and analyzed serum after intermittent PTH administration to examine the degree of oxidation. PTH administration reduced mineralization and worsened mineral and collagen quality in newly formed bone. In addition, total anti-oxidant capacity in serum was significantly decreased and the oxidative-INDEX was increased among PTH-treated compared to vehicle-treated rats, indicating serum oxidation. In conclusion, intermittent administration of PTH reduced mineral and collagen quality in newly formed mandibular bone. This effect may have been induced by oxidation.

Alveolar bone regeneration of subcutaneously transplanted rat molar.

Regeneration of alveolar bone is essential for periodontal treatment. Recently, cell replacement therapy has been focused on periodontal disease, but the source of the cells that regenerate alveolar bone is still uncertain. Therefore, to clarify the source of these bone-regenerating cells, we transplanted GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Five days after transplantation, the tooth was surrounded by connective tissue containing many blood vessels. At 10 days, bone-like tissue was formed in the connective tissue between the branches of the bifurcated root. This hard tissue expanded to almost all of this bifurcation area without osseous ankylosis after 20 days. All osteoblast-like cells in the newly formed matrix were immunopositive for GFP. In addition, these cells and the peripheral cells of the matrix showed intense immunoreactivity for BMP4, Runx2, BSP, and OPN. These results demonstrate that periodontal ligament tissue contains osteoprogenitor cells that have the ability to regenerate alveolar bone. Our model suggests that these regeneration processes might be similar to normal alveolar bone formation.

Increase of bone volume by a nanosecond pulsed laser irradiation is caused by a decreased osteoclast number and an activated osteoblasts.

The biostimulatory effects of laser irradiation focus not only in the field of soft tissue but also bone formation. Studies have shown that the light of a nanosecond pulsed laser which has a high peak power can produce stress waves in tissue. We have hypothesized that nanosecond pulsed laser irradiation stimulates bone formation. Our aim was to clarify the mechanism of increased bone volume by nanosecond pulsed laser irradiation. Rat femur was irradiated with a Q-switched Nd:YAG laser, which has a wavelength of 1064 nm. The quantification of trabecular architecture using three-dimensional morphometric analysis and measurement of bone mineral density (BMD) using pQCT was performed on day 1, day 3, day 5, and day 7 after laser irradiation. The laser effects on bone cells were also investigated using histological and immunohistochemical analysis. On day 1 after laser irradiation, bone volume (BV/TV), trabecular thickness (Tb.Th), and other parameters of the irradiated group did not significantly differ from the non-irradiation group (control). However, the mean BV/TV, Tb.Th, mineral apposition rate, and BMD of the laser group on day 7 after laser irradiation were significantly greater than those of the control. On histological analysis, the number of TRAP-positive osteoclasts was lower on day 3 after laser irradiation. Osteoblasts with activated clearance were seen in the laser irradiated group on day 1 and day 3. These data reveal that the increased bone volume by nanosecond pulsed laser irradiation causes an increase in osteoblast activity and a decrease in osteoclast number.

New 19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 analogs strongly stimulate osteoclast formation both in vivo and in vitro.

2-Methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD), an analog of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], has been shown to strongly induce bone formation both in vitro and in vivo. We have synthesized four substituents at carbon 2 of 2MD (2MD analogs), four stereoisomers at carbon 20 of the respective 2MD analogs (2MD analog-C20 isomers) and four 2MD analogs with an oxygen atom at carbon 22 (2MD-22-oxa analogs) and examined their ability to stimulate osteoclastogenesis and induce hypercalcemia. 2MD analogs were 100 times as potent as 1alpha,25(OH)2D3 in stimulating the formation of osteoclasts in vitro and in inducing the expression of receptor activator of NF-kappaB ligand (RANKL) and 25-hydroxyvitamin D3-24 hydroxylase mRNAs in osteoblasts. The osteoclast-inducing activities of 2MD analog-C20 isomers and 2MD 22-oxa analogs were much weaker than those of 2MD analogs. In addition, the activity of a 2MD analog in inducing dentine resorption was much stronger than that of 1alpha,25(OH)2D3 in the pit formation assay. Affinities to the vitamin D receptor and transcriptional activities of these compounds did not always correlate with their osteoclastogenic activities. Osteoprotegerin-deficient (OPG-/-) mice provide a suitable model for investigating in vivo effects of 2MD analogs because they exhibit extremely high concentrations of serum RANKL. The same amounts of 2MD analogs and 1alpha,25(OH)2D3 were administered daily to OPG-/- mice for 2 days. The elevation in serum concentrations of RANKL and calcium was much greater in 2MD analog-treated OPG-/- mice than in 1alpha,25(OH)2D3-treated ones. A 2MD analog was much more potent than 1alpha,25(OH)2D3 in causing hypercalcemia and in increasing soluble RANKL with enhanced osteoclastogenesis even in wild-type mice. In contrast, the administration of the 2MD analog to c-fos-deficient mice failed to induce osteoclastogenesis and hypercalcemia. These results suggest that new substituents at carbon 2 of 2MD strongly stimulate osteoclast formation in vitro and in vivo, and that osteoclastic bone resorption is indispensable for their hypercalcemic action of 2MD analogs in vivo.

Minamata disease revisited: an update on the acute and chronic manifestations of methyl mercury poisoning.

The first well-documented outbreak of acute methyl mercury (MeHg) poisoning by consumption of contaminated fish occurred in Minamata, Japan, in 1953. The clinical picture was officially recognized and called Minamata disease (MD) in 1956. However, 50 years later there are still arguments about the definition of MD in terms of clinical symptoms and extent of lesions. We provide a historical review of this epidemic and an update of the problem of MeHg toxicity. Since MeHg dispersed from Minamata to the Shiranui Sea, residents living around the sea were exposed to low-dose MeHg through fish consumption for about 20 years (at least from 1950 to 1968). These patients with chronic MeHg poisoning continue to complain of distal paresthesias of the extremities and the lips even 30 years after cessation of exposure to MeHg. Based on findings in these patients the symptoms and lesions in MeHg poisoning are reappraised. The persisting somatosensory disorders after discontinuation of exposure to MeHg were induced by diffuse damage to the somatosensory cortex, but not by damage to the peripheral nervous system, as previously believed.

Immunohistochemical study of hard tissue formation in the rat pulp cavity after tooth replantation.

While mineralized tissue is formed in the pulp cavity after tooth replantation or transplantation, little is known of this hard tissue formation. Therefore, we conducted histological and immunohistochemical evaluations of hard tissue formed in the pulp of rat maxillary molars after tooth replantation. At 5 days after replantation, degenerated odontoblasts were lining the pulp cavity. At 14 days, dentin- or bone-like tissue was present in the pulp cavity. Immunoreactivity for osteopontin (OPN) and bone sialoprotein (BSP) was strong in the bone-like tissue, but weak in the dentin-like tissue. Conversely, dentin sialoprotein (DSP) was localized in the dentin-like tissue, but not in the bone-like tissue. Cells positive for BMP4, Smad4, Runx2, and Osterix were found around the blood vessels of the root apex at 5 days. At 14 days, these cells were also localized around the bone-like tissue. Cells expressing alpha-smooth muscle actin (SMA) were seen around the newly formed bone-like tissue, whereas no such cells were found around the newly formed dentin-like tissue. In an experiment involving the transplantation of a green fluorescent protein (GFP)-transgenic rat tooth into a wild-type rat tooth socket, GFP-positive cells were detected on the surface of the bone-like tissue and over all dentin-like tissue. These results indicate that the original pulp cells had the ability to differentiate into osteoblast-like cells as well as into odontoblast-like cells.

[Methylmercury causes diffuse damage to the somatosensory cortex: how to diagnose Minamata disease].

The first acute case of methylmercury (MeHg) poisoning by the consumption of fish arose in Minamata, Japan, in 1953. It was officially recognized and called Minamata disease (MD) in 1956. There are still arguments about the definition of MD in terms of its associated clinical symptoms and lesions even 50 years after the initial recognition of MD. Studies on this MD epidemic are reviewed along with its historical background. Since MeHg dispersed from Minamata to the Shiranui Sea, residents living around the sea had been exposed to low-dose MeHg through fish consumption for about 20 years (at least from 1950 to 1968). These chronic MeHg poisoning patients complained of paresthesia at the distal parts of their extremities and around the lips even 30 years after the cessation of exposure to MeHg of anthropogenic origin. The persisting somatosensory disorders after the discontinuation of exposure to MeHg were induced by diffuse damage to the somatosensory cortex, but not by damage to the peripheral nervous system, as previously believed. Based on these findings, symptoms and lesions in MeHg poisoning are reappraised.

Treatment of OPG-deficient mice with WP9QY, a RANKL-binding peptide, recovers alveolar bone loss by suppressing osteoclastogenesis and enhancing osteoblastogenesis.

Osteoblasts express two key molecules for osteoclast differentiation, receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), a soluble decoy receptor for RANKL. RANKL induces osteoclastogenesis, while OPG inhibits it by blocking the binding of RANKL to RANK, a cellular receptor of RANKL. OPG-deficient (OPG-/-) mice exhibit severe alveolar bone loss with enhanced bone resorption. WP9QY (W9) peptide binds to RANKL and blocks RANKL-induced osteoclastogenesis. W9 is also reported to stimulate bone formation in vivo. Here, we show that treatment with W9 restores alveolar bone loss in OPG-/-mice by suppressing osteoclastogenesis and enhancing osteoblastogenesis. Administration of W9 or risedronate, a bisphosphonate, to OPG-/-mice significantly decreased the osteoclast number in the alveolar bone. Interestingly, treatment with W9, but not risedronate, enhanced Wnt/ß-catenin signaling and induced alveolar bone formation in OPG-/-mice. Expression of sclerostin, an inhibitor of Wnt/ß-catenin signaling, was significantly lower in tibiae of OPG-/-mice than in wild-type mice. Treatment with risedronate recovered sclerostin expression in OPG-/-mice, while W9 treatment further suppressed sclerostin expression. Histomorphometric analysis confirmed that bone formation-related parameters in OPG-/-mice, such as osteoblast number, osteoblast surface and osteoid surface, were increased by W9 administration but not by risedronate administration. These results suggest that treatment of OPG-/-mice with W9 suppressed osteoclastogenesis by inhibiting RANKL signaling and enhanced osteoblastogenesis by attenuating sclerostin expression in the alveolar bone. Taken together, W9 may be a useful drug to prevent alveolar bone loss in periodontitis.