Publications by authors named "Yuko Mikuni-Takagaki"

28 Publications

  • Page 1 of 1

Melatonin is a potential drug for the prevention of bone loss during space flight.

J Pineal Res 2019 Oct 19;67(3):e12594. Epub 2019 Jul 19.

Graduate School of Tokyo Medical and Dental University, Bunkyo-ku, Japan.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jpi.12594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771646PMC
October 2019

Low-Intensity Pulsed Ultrasound Prevents Development of Bisphosphonate-Related Osteonecrosis of the Jaw-Like Pathophysiology in a Rat Model.

Ultrasound Med Biol 2019 07 18;45(7):1721-1732. Epub 2019 Apr 18.

Department of Oral Science, Kanagawa Dental University Graduate School of Dentistry, Yokosuka, Japan; Joint Surgery Center, Kawasaki Saiwai Hospital, Kawasaki, Japan.

We developed a rat model of bisphosphonate-related osteonecrosis of the jaw (BRONJ) by removing a maxillary molar tooth (M1) from ovariectomized rats after treatment with alendronate. To mimic periodontitis, some of the rats were administered Porphyromonas gingivalis (p. gingivalis) at the M1 site every 2 to 3 d for 2 wk. Rats pretreated with alendronate plus p. gingivalis showed delayed healing of socket epithelia, periosteal reaction of alveolar bone formation and lower bone mineral density in the alveolus above adjacent M2 teeth. These abnormalities were prevented by tooth socket exposure to 20 min/d low-intensity pulsed ultrasound (LIPUS), which restored diminished expression of RANKL, Bcl-2, IL-6, Hsp70, NF-κB and TNF-α messenger ribonucleic acids in remote bone marrow, suggesting LIPUS prevented development of BRONJ-like pathophysiology in rat by inducing systemic responses for regeneration, in addition to accelerating local healing. Non-invasive treatment by LIPUS, as well as low-level laser therapy, may be useful for medication-related osteonecrosis of the jaw patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ultrasmedbio.2019.02.015DOI Listing
July 2019

Correction of Mass Spectrometric Interferences for Rapid and Precise Isotope Ratio Measurements of Calcium from Biological Samples Using ICP-Mass Spectrometry.

Anal Sci 2019 Jul 5;35(7):793-798. Epub 2019 Apr 5.

Geochemical Research Center, The University of Tokyo.

Stable isotope compositions of calcium (Ca) provide useful information concerning metabolic alterations of Ca in human and animal bodies. For the measurements of Ca isotope ratio, great care must be taken for the mass spectrometric interferences on Ca isotopes (Ca, Ca, and Ca) from doubly charged strontium (Sr) ions (Sr, Sr, and Sr). To obtain reliable stable isotope data of Ca, we developed a new correction technique for the mass spectrometric interferences by Sr ions based on standard addition method. Addition of a small fraction of Sr onto a Ca solution shifts the measured Ca isotope ratios on a three-isotope diagram (i.e., δCa and δCa) along a mixing line defined by both the true Ca isotope ratio and the Sr isotope ratio. Therefore, the true Ca isotope ratio of a sample can be obtained as the crossover point of mass dependent fractionation line and the mixing line. With the present correction technique, precise and accurate isotope ratio measurements can be made on analyte solutions having a C/C ratio (concentration ratio) of 0.03, which is 6 times higher than the C/C ratio applicable to the conventional correction technique.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2116/analsci.18P440DOI Listing
July 2019

In-office bleaching for the remineralization of enamel lesions filled with organic components of red wine.

Am J Dent 2018 Feb;31(1):13-16

Department of Oral Interdisciplinary Medicine, Graduate School of Dentistry, Kanagawa Dental University, Kanagawa, Japan.

Purpose: To investigate the effects of in-office bleaching on the remineralization of enamel lesions filled with organic components of red wine.

Methods: Enamel specimens were exposed to 0.1% NaF solution for 1 minute immersed in red wine for 5 days at 37°C, and subjected to in-office bleaching followed by remineralization in 1.5 mM CaCl₂, 0.9 mM KH₂PO₄, 130 mM KCl, 20 mM HEPES, pH 7.0, at 37°C for 28 days. The presence of organic substances on the enamel surface was detected by Raman spectroscopy. The specimens were also subjected to transverse microradiography (TMR).

Results: Raman spectroscopy of baseline lesions showed characteristic peaks at 1,300-1,600 cm-1 which disappeared in bleached specimens. TMR showed that red wine formed subsurface lesions with surface content at approximately 22 mineral volume %. The integrated mineral loss (IML) was significantly lower in unbleached remineralized specimens than at baseline (P< 0.05). The IML of bleached remineralized specimens was lower than that of unbleached specimens, although not significantly (P> 0.05). Lesion depth was significantly lower in the bleached than in the unbleached group (P< 0.05).

Clinical Significance: In-office bleaching can enhance the remineralization of enamel lesions filled with organic components of red wine.
View Article and Find Full Text PDF

Download full-text PDF

Source
February 2018

13. Effect of Low-Intensity Pulsed Ultrasound (LIPUS) on Remote Bone Marrow in Rats With Healing Socket.

J Orthop Trauma 2016 Aug;30(8):S5-6

*Department of Oral Function and Restoration, Division of Periodontology; †Department of Oral Sciences; ‡Department of Neuroanatomy, Histology and Embryology; §Department of Oral and Maxillofacial Rehabilitation; ‖Department of Radiopraxis Science; ¶Department of Oral Sciences, Divisions of Dentistry of Circulation Control; and **Molecular and Cellular Biology of Mineralized Tissues, Kanagawa Dental University Graduate School of Dentistry.

Objective: We reported at the previous annual meeting that LIPUS treatment of the molar tooth sockets of retired breeder rats accelerated alveolar bone healing, and that associated humoral effects were seen with elevated blood flow. Namely, LIPUS induced VEGF/angiogenesis along with elevated baseline blood flow rate, which was further associated with a sudden depression of blood flow rate in the socket immediately after cessation of LIPUS treatment. Prior injection with EP4 PGE2 receptor antagonist, but not EP3 antagonist, abolished this LIPUS-induced depression, and topical application of PGE2 to the socket epithelium mimicked the LIPUS-induced depression. In fact, the serum level of PGE2 increased after LIPUS treatment, and significantly increased in the blood flow rate at remote sites on the foot dorsum and tail after 20 minutes. Therefore, in the current study, we examined the tibia bone marrow, which is likely to respond to circulating PGE2.

Methods: Right maxillary first molars were removed from retired female breeder rats in both the LIPUS and the control groups. LIPUS was applied extrabuccally to the socket every 24 hours for 2 weeks starting one day after extraction. Removed bone samples were fixed with 4% formaldehyde to prepare undecalcified frozen sections using Kawamoto's method for immunohistochemical or histochemical staining. Bone marrow samples dissected from the tibia were treated with RNAlater (Ambion) for later RT-PCR analysis.

Results And Discussion: Chemokine receptor CXCR4-positive bone marrow cells increased in the tibia of the LIPUS-treated rat. Together with ubiquitously expressed CXCL12(SDF-1), it is suggested that PGE2 released from the exposed socket is responsible for the recruitment, proliferation and mobilization of the precursors of bone forming cells. LIPUS is thought to exert humoral effects by recruiting bone marrow cells into the healing socket along with VEGF/angiogenesis induced by PGE2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.bot.0000489986.43352.faDOI Listing
August 2016

9. Effects of Low-Intensity Pulsed Ultrasound (LIPUS) on Osteoclasts and Osteoblasts: Analysis Using an Assay System With Fish Scale as a Model of Bone.

J Orthop Trauma 2016 Aug;30(8):S4

*Kanazawa University; †Okayama University; ‡Toyama Prefectural University; §University of Toyama; ‖Kanagawa Dental University; and ¶Tokyo Medical and Dental University.

Objective: Low-intensity pulsed ultrasound (LIPUS) provides noninvasive therapeutic treatment to accelerate fracture repair and distraction osteogenesis. However, most studies concerning the influence of LIPUS on bone metabolism have been conducted in vivo systems using osteoblastic cells. Therefore, details of the direct effect of LIPUS on osteoclasts are not yet fully understood. Teleost scale is a calcified tissue that contains osteoclasts and osteoblasts. Its bone matrix consists of type I collagen and hydroxyapatite, and is similar to that of mammalian bone. Therefore, we examined the effect of LIPUS on the osteoclasts and osteoblasts of zebrafish and goldfish scales, as a model of the bone matrix simplified to its bare bones.

Methods: Ultrasound was generated with the Sonic Accelerated Fracture Healing System (SAFHS 4000J; Teijin Pharma, Ltd). Scales were collected from zebrafish under anesthesia; they were then treated with LIPUS for 20 minutes, incubated at 15°C for 3, 6, and 18 hours in L-15 medium, and subjected to measurement of the mRNA expression. Following the osteoclast induction by the autotransplantation of goldfish scales, we further examined the number of apoptotic osteoclasts after LIPUS treatment.

Results And Discussion: At 3 hours of incubation after LIPUS treatment, osteoclastic marker expression decreased while osteoblastic markers increased. Using GeneChip analysis of zebrafish scales treated by LIPUS, we found that cell death-related genes were up-regulated by LIPUS treatment. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was elevated by treatment with LIPUS at 3 hours of incubation. Thus, we conclude that LIPUS promotes apoptosis in osteoclasts shortly after exposure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.bot.0000489982.40329.52DOI Listing
August 2016

5. Accelerated Fracture Healing Targeting Periosteal Cells: Possibility of Combined Therapy of Low-Intensity Pulsed Ultrasound (LIPUS), Bone Graft, and Growth Factor (bFGF).

J Orthop Trauma 2016 Aug;30(8):S3

*Department of Orthopaedic Surgery, Kitasato University School of Medicine; †Department of Orthopaedic Surgery, Kitasato University Medical Center; and ‡Molecular and Cellular Biology of Mineralized Tissues, Kanagawa Dental University Graduate School of Dentistry.

We have studied the mechanism of fracture healing, and the effect of LIPUS, bone graft and growth factor on accelerating fracture healing. We present here the results of our research. To examine callus formation cells in fracture healing, we made marrow GFP chimera mice and a fracture model of marrow mesenchymal stem cell GFP chimera mice. It was demonstrated that periosteal cells were essential for callus formation. We focused on periosteal cells and examined the effect of LIPUS. In an in vitro experiment using a cultured part of the femur, LIPUS promoted ossification of the periosteal tissue. Further, LIPUS accelerated VEGF expression in the experiment using the femoral fracture model of mice. From these results, it was suggested that activation of periosteal cells might play a role in the fracture healing mechanism of LIPUS. Next, we discussed the possibility of combined therapy of LIPUS, bone graft and growth factor. Therapy involving the topical administration of bFGF using a controlled release system and bone graft could promote callus formation. In addition, LIPUS was able to promote membranaceous ossification after the bone graft. It was suggested that combined therapy of LIPUS, bone graft and bFGF could be a new option for treating fractures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.bot.0000489990.66226.51DOI Listing
August 2016

Mode & mechanism of low intensity pulsed ultrasound (LIPUS) in fracture repair.

Ultrasonics 2016 08 9;70:45-52. Epub 2016 Apr 9.

Kanagawa Dental University, Graduate School of Dentistry, Yokosuka, Japan.

It has been 30years since the first level one clinical trial demonstrated low intensity pulsed ultrasound (LIPUS) could accelerate fracture repair. Since 1994 numerous investigations have been performed on the effect of LIPUS. The majority of these studies have used the same signal parameters comprised of an intensity of 30mW/cm(2) SATA, an ultrasound carrier frequency of 1.5MHz, pulsed at 1kHz with an exposure time of 20minutes per day. These studies show that a biological response is stimulated in the cell which produces bioactive molecules. The production of these molecules, linked with observations demonstrating the enhanced effects on mineralization by LIPUS, might be considered the general manner, or mode, of how LIPUS stimulates fractures to heal. We propose a mechanism for how the LIPUS signal can enhance fracture repair by combining the findings of numerous studies. The LIPUS signal is transmitted through tissue to the bone, where cells translate this mechanical signal to a biochemical response via integrin mechano-receptors. The cells enhance the production of cyclo-oxygenese 2 (COX-2) which in turn stimulates molecules to enhance fracture repair. The aim of this review is to present the state of the art data related to LIPUS effects and mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ultras.2016.03.016DOI Listing
August 2016

Low-intensity pulsed ultrasound induces apoptosis in osteoclasts: Fish scales are a suitable model for the analysis of bone metabolism by ultrasound.

Comp Biochem Physiol A Mol Integr Physiol 2016 May 2;195:26-31. Epub 2016 Feb 2.

Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan.

Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3h of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal-less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18h of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in osteoclasts using the TUNEL (TdT-mediated dUTP nick end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3h of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbpa.2016.01.022DOI Listing
May 2016

Chemical alteration by tooth bleaching of human salivary proteins that infiltrated subsurface enamel lesions--experimental study with bovine lesion model systems.

Dent Mater J 2014 ;33(5):663-8

Department of Cariology and Restorative Dentistry, Graduate School of Dentistry, Kanagawa Dental University.

Salivary macromolecules infiltrate white and brown spot enamel lesions and adsorb onto hydroxyapatite. Calcium-binding salivary proteins such as statherin hinder remineralization of these lesions. We assessed whether bleaching agents can remove salivary components that have infiltrated and bound to experimental subsurface lesions in bovine enamel prepared by immersing specimens in acid and then human saliva. Transversal microradiography showed that such demineralized lesions mimicked incipient carious lesions. Bound proteins to the experimental and untreated control specimens were eluted in a stepwise manner with phosphatebuffered saline, 0.4 M phosphate buffer, and 1 M HCl. SDS-PAGE of dialyzed extracts showed that specific salivary proteins bound to the lesions, while virtually no protein bands were detected if the specimens were bleached. Western blotting showed that even statherin, which was more firmly bound than other proteins, was removed. In-office bleaching agent may be useful in treating enamel lesions for removing proteins bound to these lesions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4012/dmj.2014-046DOI Listing
September 2016

A not-so-systematic review.

Can J Surg 2014 Oct;57(5):E150-1

Project Professor, Division of Molecular and Cellular Biology of Mineralized Tissues, Department of Oral Sciences, Kanagawa Dental University, Graduate School of Dentistry, Yokosuka, Japan.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183692PMC
http://dx.doi.org/10.1503/cjs.009514DOI Listing
October 2014

Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

PLoS One 2014 7;9(4):e93840. Epub 2014 Apr 7.

Department of Bone and Mineral Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka, Japan.

Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH)2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH)2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093840PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977859PMC
January 2015

Zebrafish scales respond differently to in vitro dynamic and static acceleration: analysis of interaction between osteoblasts and osteoclasts.

Comp Biochem Physiol A Mol Integr Physiol 2013 Sep 28;166(1):74-80. Epub 2013 Apr 28.

Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0942, Japan.

Zebrafish scales consist of bone-forming osteoblasts, bone-resorbing osteoclasts, and calcified bone matrix. To elucidate the underlying molecular mechanism of the effects induced by dynamic and static acceleration, we investigated the scale osteoblast- and osteoclast-specific marker gene expression involving osteoblast-osteoclast communication molecules. Osteoblasts express RANKL, which binds to the osteoclast surface receptor, RANK, and stimulates bone resorption. OPG, on the other hand, is secreted by osteoblast as a decoy receptor for RANKL, prevents RANKL from binding to RANK and thus prevents bone resorption. Therefore, the RANK-RANKL-OPG pathway contributes to the regulation of osteoclastogenesis by osteoblasts. Semaphorin 4D, in contrast, is expressed on osteoclasts, and binding to its receptor Plexin-B1 on osteoblasts results in suppression of bone formation. In the present study, we found that both dynamic and static acceleration at 3.0×g decreased RANKL/OPG ratio and increased osteoblast-specific functional mRNA such as alkaline phosphatase, while static acceleration increased and dynamic acceleration decreased osteoclast-specific mRNA such as cathepsin K. Static acceleration increased semaphorin 4D mRNA expression, while dynamic acceleration had no effect. The results of the present study indicated that osteoclasts have predominant control over bone metabolism via semaphorin 4D expression induced by static acceleration at 3.0×g.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbpa.2013.04.023DOI Listing
September 2013

Multimolecular salivary mucin complex is altered in saliva of cigarette smokers: detection of disulfide bridges by Raman spectroscopy.

Biomed Res Int 2013 26;2013:168765. Epub 2012 Dec 26.

Department of Functional Biology, Kanagawa Dental College, 82 Inaokacho, Yokosuka 238-8580, Japan.

Saliva contains mucins, which protect epithelial cells. We showed a smaller amount of salivary mucin, both MG1 and MG2, in the premenopausal female smokers than in their nonsmoking counterparts. Smokers' MG1, which contains almost 2% cysteine/half cystine in its amino acid residues, turned out to be chemically altered in the nonsmoker's saliva. The smaller acidic glycoprotein bands were detectable only in smoker's saliva in the range of 20-25 kDa and at 45 kDa, suggesting that degradation, at least in part, caused the reduction of MG1 mucin. This is in agreement with the previous finding that free radicals in cigarette smoke modify mucins in both sugar and protein moieties. Moreover, proteins such as amylase and albumin are bound to other proteins through disulfide bonds and are identifiable only after reduction with DTT. Confocal laser Raman microspectroscopy identified a disulfide stretch band of significantly stronger intensity per protein in the stimulated saliva of smokers alone. We conclude that the saliva of smokers, especially stimulated saliva, contains significantly more oxidized form of proteins with increased disulfide bridges, that reduces protection for oral epithelium. Raman microspectroscopy can be used for an easy detection of the damaged salivary proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2013/168765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591210PMC
November 2013

Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales.

Zoolog Sci 2013 Mar;30(3):217-23

Japan Aerospace Exploration Agency, Tsukuba, Ibaraki 305-8505, Japan.

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-κB ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2108/zsj.30.217DOI Listing
March 2013

Disruption of NF-κB1 prevents bone loss caused by mechanical unloading.

J Bone Miner Res 2013 Jun;28(6):1457-67

Division of Molecular Signaling and Biochemistry, Department of Health Improvement, Kyushu Dental University, Fukuoka, Japan.

Mechanical unloading, such as in a microgravity environment in space or during bed rest (for patients who require prolonged bed rest), leads to a decrease in bone mass because of the suppression of bone formation and the stimulation of bone resorption. To address the challenges presented by a prolonged stay in space and the forthcoming era of a super-aged society, it will be important to prevent the bone loss caused by prolonged mechanical unloading. Nuclear factor κB (NF-κB) transcription factors are activated by mechanical loading and inflammatory cytokines. Our objective was to elucidate the role of NF-κB pathways in bone loss that are caused by mechanical unloading. Eight-week-old wild-type (WT) and NF-κB1-deficient mice were randomly assigned to a control or mechanically unloaded with tail suspension group. After 2 weeks, a radiographic analysis indicated a decrease in bone mass in the tibias and femurs of the unloaded WT mice but not in the NF-κB1-deficient mice. An NF-κB1 deficiency suppressed the unloading-induced reduction in bone formation by maintaining the proportion and/or potential of osteoprogenitors or immature osteoblasts, and by suppression of bone resorption through the inhibition of intracellular signaling through the receptor activator of NF-κB ligand (RANKL) in osteoclast precursors. Thus, NF-κB1 is involved in two aspects of rapid reduction in bone mass that are induced by disuse osteoporosis in space or bed rest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.1866DOI Listing
June 2013

Differential age-related bone architecture changes between female and male STR/Ort mice.

Exp Anim 2012 ;61(1):59-66

Department of Orthopedic Surgery, Kitasato University School of Medicine, Kanagawa, Japan.

The incidence of spontaneous osteoarthritis (OA) in female STR/Ort mice is much lower than that observed in male STR/Ort mice; however, the reason for the differential incidence of OA between sexes has not been elucidated. Here, we investigated and compared age- and sex-related bone mineral density and architectural changes in male and female STR/Ort mice. Bone architecture and bone mineral density (BMD) of femurs were examined in 5-, 10-, 15-, 20-, and 35-week-old male and female STR/Ort mice by microscopic computed tomography (µCT). Angular degrees of internal tibial torsion (ADITT) were also measured in mice at 5, 15, and 35 weeks of age. Earlier decreases of cancellous volume and BMD were found in male STR/Ort mice. Using µCT, an age-related decline of bone marrow space in femoral diaphysis was observed in both males and females but was more dramatic in females. In addition, an earlier increase of ADITT was observed in male STR/Ort mice, suggesting that internal rotation of the tibia may contribute to OA. Age- and sex-related bone architectural changes clearly differ between male and female STR/Ort mice. These differences in bone structure, particularly ADITT, may explain the differential incidence of OA in STR/Ort mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1538/expanim.61.59DOI Listing
May 2012

Low intensity pulsed ultrasound accelerates delayed healing process by reducing the time required for the completion of endochondral ossification in the aged mouse femur fracture model.

Exp Anim 2011 ;60(4):385-95

Department of Orthopedic Surgery, Kitasato University School of Medicine, 1–15–1 Kitasato, Sagamihara Minami-ku, 252-0374 Kanagawa, Japan.

The aim of this study is to clarify the effect of low intensity pulsed ultrasound (LIPUS) on shortening of the fracture healing period and endochondral ossification during the fracture healing process. We first established a model of aging-related delayed union fractures consisting of aged mouse (C57BL/6J; 40 weeks old) with closed femur fractures. We compared the healing process of 40-week-old mice to the healing process of 8-week-old (young) mice using radiological and histological analysis. In aged mice, some cartilage formation was observed 10 days after the fracture; however, endochondral ossification and hard callus bridging were observed 21 and 28 days after the fracture, respectively, whereas cartilage remained in the callus on day 28, suggesting delayed endochondral ossification following bone remodeling. Meanwhile, in aged mice with LIPUS treatment, cartilage formation was similar to that in aged mice without LIPUS; however, hard callus bridging and bone remodeling were observed 21 and 28 days after fracture, respectively, suggesting that LIPUS shortened the healing period due to promotion of endochondral ossification. Immunohistochemical analysis showed marked expression of vascular endothelial growth factor and neovascularization in the fibrous tissue comprising the periosteum that surrounded the whole callus. A cell migration test involving primary cultured human endothelial cells also showed promotion of cell migration by LIPUS. These results suggested that endothelial cell migration and neovascularization, which were observed around fracture sites, played a part in the mechanism of promotion of endochondral ossification by LIPUS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1538/expanim.60.385DOI Listing
November 2011

A novel underuse model shows that inactivity but not ovariectomy determines the deteriorated material properties and geometry of cortical bone in the tibia of adult rats.

J Bone Miner Metab 2011 Jul 3;29(4):422-36. Epub 2010 Dec 3.

Division of Biochemistry and Molecular Biology, Department of Functional Biology, Kanagawa Dental College, 82 Inaokacho, Yokosuka 238-8580, Japan.

Our goal in this study was to determine to what extent the physiologic consequences of ovariectomy (OVX) in bones are exacerbated by a lack of daily activity such as walking. We forced 14-week-old female rats to be inactive for 15 weeks with a unique experimental system that prevents standing and walking while allowing other movements. Tibiae, femora, and 4th lumbar vertebrae were analyzed by peripheral quantitative computed tomography (pQCT), microfocused X-ray computed tomography (micro-CT), histology, histomorphometry, Raman spectroscopy, and the three-point bending test. Contrary to our expectation, the exacerbation was very much limited to the cancellous bone parameters. Parameters of femur and tibia cortical bone were affected by the forced inactivity but not by OVX: (1) cross-sectional moment of inertia was significantly smaller in Sham-Inactive rat bones than that of their walking counterparts; (2) the number of sclerostin-positive osteocytes per unit cross-sectional area was larger in Sham-Inactive rat bones than in Sham-Walking rat bones; and (3) material properties such as ultimate stress of inactive rat tibia was lower than that of their walking counterparts. Of note, the additive effect of inactivity and OVX was seen only in a few parameters, such as the cancellous bone mineral density of the lumbar vertebrae and the structural parameters of cancellous bone in the lumbar vertebrae/tibiae. It is concluded that the lack of daily activity is detrimental to the strength and quality of cortical bone in the femur and tibia of rats, while lack of estrogen is not. Our inactive rat model, with the older rats, will aid the study of postmenopausal osteoporosis, the etiology of which may be both hormonal and mechanical.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00774-010-0241-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3132588PMC
July 2011

Repeated freeze-thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons.

Cell Tissue Bank 2012 Mar 30;13(1):71-80. Epub 2010 Nov 30.

Department of Orthopaedic Surgery, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan.

Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze-thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze-thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze-thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze-thaw cycles. In patella tendons, the ultimate stress, Young's modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze-thaw cycles. In conclusion, we identified that cells surviving after freeze-thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze-thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10561-010-9234-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286509PMC
March 2012

Prolonged endochondral bone healing in senescence is shortened by low-intensity pulsed ultrasound in a manner dependent on COX-2.

Ultrasound Med Biol 2010 Jul;36(7):1098-108

Department of Orthopedic Surgery, Kitasato University School of Medicine, Sagamihara, Japan.

To test whether mechanical loading produces faster healing in aged mice, fractured femurs of aged 1-year-old mice were subjected to low-intensity pulsed ultrasound (LIPUS), a treatment that is routinely used to help heal fractures in humans. Cyclooxygenase-2 knockout mice (COX-2(-/-)), which lack an immediate early mediator of mechanical stimulation, were also studied by histochemistry, microcomputed tomography and quantitative polymerase chain reaction to determine the role of COX-2. The healing in the aged COX-2(-/-) mice is slow during the endochondral bone remodeling (>30 d), a period generally prolonged in senescence. For aged wild-type mice, LIPUS halved the endochondral phase to about 10 d, whereas that was not the case for aged COX-2(-/-) mice, which showed no apparent shortening of the prolonged endochondral-phase healing time. Injecting prostaglandin E(2) receptor agonists, however, rescued the COX-2(-/-) callus from insensitivity to LIPUS. In conclusion, COX-2 is a limiting factor in the delayed endochondral bone healing and is induced by LIPUS, which normalizes healing rate to the wild-type level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ultrasmedbio.2010.04.011DOI Listing
July 2010

Osteoarthritic changes of the patellofemoral joint in STR/OrtCrlj mice are the earliest detectable changes and may be caused by internal tibial torsion.

Connect Tissue Res 2009 ;50(4):243-55

Department of Orthopaedic Surgery, Kitasasto University School of Medicine, Kanagawa, Japan.

STR/ort mice develop a naturally occurring osteoarthritis (OA) of the knee joints. However, the evaluation of early OA changes has been difficult due to variability caused by gender, individual differences, and differences between the right and left lower limbs. The objective of this study was to analyze the variability of the early OA changes with age in STR/ort mice and to identify the cause of onset. A total of 115 STR/OrtCrlj mice aged 10-45 weeks were examined. In addition to conventional radiological and histological evaluation of the knee joints, histological sections were used to examine the patellofemoral, femorotibial, and growth plate cartilage under similar conditions. A morphological evaluation of tibiae, including micro-3-dimensional computed tomography, was performed. Radiological evaluation showed OA changes in the joints of mice over 35 weeks old and histological evaluation showed early OA changes in the femorotibial joints of mice over 26 weeks old. However, these changes were not common in all individuals. In contrast, most common and reproducible OA changes were observed in the bilateral patellofemoral joints of all individuals, and even in subjects ranging from 10 to 20 weeks of age. Morphological evaluations also demonstrated an abnormal tibial internal torsion that increased with age and was associated with medial patellar dislocation. In conclusion, the earliest histological OA change was observed in the patellofemoral joint prior to similar observations in the femorotibial joint. Internal tibial torsion may be a cause of OA in the patellofemoral joints, which leads to the development of medial femorotibial OA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/03008200902836065DOI Listing
October 2009

Therapeutic ultrasound induces periosteal ossification without apparent changes in cartilage.

Connect Tissue Res 2009 ;50(1):55-63

Department of Orthopedic Surgery, Kitasato University School of Medicine, Sagamihara, Japan.

Low intensity pulsed ultrasound (LIPUS) is an extremely useful noninvasive treatment which halves the duration of fracture healing when the bone is exposed once a day for 20 min. To elucidate the direct reactions of bone and cartilage, dissected rat femora were immobilized in culture dish wells, exposed to LIPUS from a certain angle every day, and the local pattern of ossification was analyzed in relation to the ultrasound. Daily 20-min exposures were started 24 hr after isolation of the femora, and at days 5, 10, and 15, samples were harvested for measurements, morphological, and histochemical analyses. While the gross features of the samples were identical to the untreated controls, extended mineralization of the periosteum was observed with alizarin red staining, antiosteocalcin immunohistochemical staining, and micro-three dimensional computed tomography. Interestingly, the newly deposited mineral was found perpendicular to the ultrasound path, strongly suggesting that LIPUS accelerates periosteal bone formation. Zones of epiphyseal cartilage and hypertrophic and calcified cartilage did not exhibit any differences with and without this exposure. LIPUS also did not influence the secreted proteoglycan components or amounts in the culture medium. The absence of any additional longitudinal growth of the femur demonstrated that LIPUS did not accelerate endochondral bone formation. We conclude that cartilage alone does not directly respond to therapeutic ultrasound, whereas the periosteum does.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/03008200802419855DOI Listing
April 2009

Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study.

Arthritis Res Ther 2008 11;10(4):R77. Epub 2008 Jul 11.

Department of Orthopaedic Surgery, Yokohama City University School of Medicine, Yokohama City, Kanagawa, Japan.

Introduction: The effect of low-intensity pulsed ultrasound (LIPUS) on cell growth was examined in three-dimensional-cultured chondrocytes with a collagen sponge. To elucidate the mechanisms underlying the mechanical activation of chondrocytes, intracellular signaling pathways through the Ras/mitogen-activated protein kinase (MAPK) and the integrin/phosphatidylinositol 3 kinase (PI3K)/Akt pathways as well as proteins involved in proliferation of chondrocytes were examined in LIPUS-treated chondrocytes.

Methods: Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly sacrificed pigs. Isolated chondrocytes mixed with collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. Experimental cells were cultured with daily 20-minute exposures to LIPUS. The chondrocytes proliferated and a collagenous matrix was formed on the surface of the sponge. Cell counting, histological examinations, immunohistochemical analyses and western blotting analysis were performed.

Results: The rate of chondrocyte proliferation was slightly but significantly higher in the LIPUS group in comparison with the control group during the 2-week culture period. Western blot analysis showed intense staining of type-IX collagen, cyclin B1 and cyclin D1, phosphorylated focal adhesion kinase, and phosphorylated Akt in the LIPUS group in comparison with the control group. No differences were detected, however, in the MAPK, phosphorylated MAPK and type-II collagen levels.

Conclusion: LIPUS promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a three-dimensional culture using a collagen sponge. In addition, the anabolic LIPUS signal transduction to the nucleus via the integrin/phosphatidylinositol 3-OH kinase/Akt pathway rather than the integrin/MAPK pathway was generally associated with cell proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/ar2451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575623PMC
February 2009

AlphaVbeta3 integrin ligands enhance volume-sensitive calcium influx in mechanically stretched osteocytes.

J Bone Miner Metab 2006 ;24(6):498-504

National Hyogo Chuo Hospital, 1314 Ohara, Sanda, Hyogo, 669-1515, Japan.

We propose that specific osteocyte-matrix interactions regulate the volume-sensitive calcium influx pathway, which we have shown is mediated by stretch-activated cation channels (SA-Cat) and is essential for the stretch-activated anabolic response in bone. The current study measured the hypotonic swelling-induced increase in cytosolic calcium concentration, [Ca(2+)](i), in rat osteocytes, and found that cells adherent to different matrices behave differently. Osteopontin and vitronectin, matrix molecules that bind the alpha(V)beta(3) integrin, induced larger responses to the hypotonic swelling than other matrix molecules that bind other integrins. Addition of echistatin, which is a soluble alpha(V)beta(3) ligand, significantly enhanced the hypotonic [Ca(2+)](i) increase in addition to inducing an immediate increase in [Ca(2+)](i) by itself. These results strongly support the contention that alpha(V)beta(3) integrin signaling in osteocytes interacts with that in mechanotransduction, which is downstream of SA-Cat.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00774-006-0716-xDOI Listing
March 2007

A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism.

J Biol Chem 2006 Nov 7;281(44):33814-24. Epub 2006 Sep 7.

Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.

Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2(-/-) mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2(-/-) mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2(-/-) mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2(-/-) calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2(-/-) calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M607290200DOI Listing
November 2006

Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation.

Bone 2004 Oct;35(4):850-8

Department of Orthopedic Surgery, Kitasato University School of Medicine, Sagamihara, Japan.

Mesenchymal stem cells (MSCs) are thought to be multipotential, capable of differentiating into multiple lineages. We attempted to characterize rat cells derived from fetal circulating blood (FCBCs) that displayed a fibroblastic morphology and differentiated into osteoblastic and chondrocytic lineages. Notably, they differentiated into a chondrocyte-specific phenotype on plastic culture dishes in medium supplemented only with 10% fetal bovine serum (FBS) without the use of a three-dimensional culture substrate. Bone marrow-derived cells did not convey such phenotypic expression under the same conditions. The characteristic features of these cells were analyzed by reverse transcription polymerase chain reaction, immunohistological and von Kossa staining, and by immuno-dot blotting. In one population, expression of collagen types II and X was detected in differentiated cells at the same levels as observed in chondrocytes derived from rat rib cartilage. In another population, parathyroid hormone receptor, alkaline phosphatase, and osteocalcin were also expressed at levels almost equal to those observed in long bone-derived osteoblasts. After 3 weeks in culture, extensively condensed cell masses, stained with anti-type II collagen antibody, could be distinguished histologically from small, multilayered, von Kossa-positive nodules, which stained with anti-osteocalcin, but not with anti-type II collagen antibody. In addition, the FCBCs differentiated into adipogenic cells in the presence of methyl-isobutyl xanthine, dexamethasone, insulin, and indomethacin. These cells expressed PPARgamma2 mRNA and accumulated lipid vesicles detectable by Oil red-O staining. Our findings suggest that FCBCs have the potential to readily differentiate into multiple lineages and that they are distinct from mesenchymal stem cells derived from bone marrow or circulating blood from more mature and adults in their spontaneous differentiation in the absence of specific factors such as transforming growth factor-beta (TGF-beta) or dexamethasone, or a three-dimensional culture environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2004.05.006DOI Listing
October 2004

Distinct anabolic response of osteoblast to low-intensity pulsed ultrasound.

J Bone Miner Res 2003 Feb;18(2):360-9

Department of Orthopedic Surgery, Kitasato University School of Medicine, Sagamihara, Japan.

Low-intensity pulsed ultrasound, a form of mechanical energy transmitted as high-frequency acoustical pressure waves, provides noninvasive therapeutic treatment for accelerating fracture repair and distraction osteogenesis. Relatively young osteoblasts respond to ultrasound by transiently upregulating message levels of immediate-early genes as well as that of osteocalcin and insulin-like growth factor I (IGF-I). Osteocytes derived from newborn rat tibia and calvaria responded to a lesser extent only in c-fos and cyclooxygenase-2 (COX-2) messages. Compared with the stretched osteocytes, which use stretch-activated and parathyroid hormone (PTH)-potentiated Ca2+ influx as an entry route to the protein kinase A (PKA) signal transduction pathways, there was no evidence of Ca2+ internalization by any of the cells tested on exposure to the ultrasound. On the other hand, inhibitors of p38 mitogen-activated protein kinase (MAPK) and upstream phosphoinositide 3-kinase (PI3K) blocked COX-2 and osteocalcin upregulation by the ultrasound-exposed ST2, murine bone marrow-derived cells. This is distinct from the aforementioned osteocytic response to low-frequency stretching and implies the involvement of integrins. Our findings suggested that accelerated fracture repair and distraction osteogenesis by the low-intensity pulsed ultrasound depend, at least in part, on the stimulation of osteoblastic cells at relatively early stages of osteogenic lineage. Bone is under control of multiple regulatory mechanisms so that diverse physical forces can be reflected to the microenvironment of each cell, in turn, to the entire bone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1359/jbmr.2003.18.2.360DOI Listing
February 2003