The 18th Annual Meeting of the Society for Hard Tissue Regenerative Biology
in Tokushima Bunri University

Chairman: Makoto Arisue

Clinical importance of regeneration or reconstruction medicine for hard tissues in aging societies.

Manabu Ito

Dept. of Orthopaedic Surgery, Hokkaido University Graduate School of Medicine.

@Japan is now moving to a super-aging society with the highest speed in the world. Population statistics show that more than 40% of the population in Japan will exceed 65 years of age by the middle of this century. In order to maintain the quality of life of the elderly, regeneration or reconstruction medicine of hard tissue is becoming of highest clinical importance. Through my clinical experience treating senile patients with osteoporotic spinal fractures and spinal deformities, I would like to introduce the current status of surgical treatment for spinal fractures in osteoporotic collapsing spine and to discuss the future directions of treatment of human hard tissues.		 Effects of Carbonic Anhydrase Inhibitor acetazolamide (AZ) on osteoclasts and bone texture

Chihiro Shinohara1), Fumiaki Kawano1) and Kikuji Yamashita2)

1)Comprehensive Dentistry1), Institute of Health Biosciences, The University of Tokushima Graduate School
2)Oral and Maxillofacial Anatomy, Institute of Health Biosciences, The University of Tokushima Graduate School

Osteoclast-targeting pharmaceutical products have been developed for the treatment of abnormal bone metabolism. We investigated the effects of carbonic anhydrase inhibitor acetazolamide (AZ) on osteoclasts to design a new drug. Bone resorption activity and the number of osteoclasts decreased by AZ in a doze dependent manner, showing morphological apoptotic characteristics in bone marrow culture. The area of trabecular bone was increased and the density of osteoclasts was decreased in the AZ-treated rats. These results suggested that AZ is useful for regulation of bone metabolism as pharmaceutical products.
Regeneration induction therapy based on functional biomaterials with growth factor delivery

Masaya Yamamoto and Yasuhiko Tabata

Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University

Tissue engineering is a newly emerging biomedical form to create a local environment which enables stem cells to promote their proliferation and differentiation for induction of tissue regeneration. Growth factor is a biosignaling protein that can regulate those cell functions in the local environment to regulate and accelerate tissue regeneration. For the successful growth factor-induced tissue regeneration, it is important to develop functional biomaterials which can function not only as a scaffold to support the proliferation and differentiation of stem cells but also as a drug delivery system (DDS) to achieve the appropriate timing and concentration of growth factors at a body site to be regenerated. This paper overviews the recent development and clinical application of functional biomaterials with growth factor delivery necessary to realize the induction therapy of tissue regeneration.		 Development of apatite-fiber scaffolds and their application to bone regeneration

Mamoru Aizawa1) and Morio Matsumoto2)

1)Department of Applied Chemistry, School of Science and Technology, Meiji University
2)Department of Orthopaedic Surgery, School of Medicine, Keio University

We have successfully developed the apatite-fiber scaffolds (AFS) with inter-connected pores of 100-250 ?m for tissue engineering of bone. The cells three-dimensionally cultured using the present scaffolds indicated higher cell activities, such as cell proliferation and differentiation than the two-dimensionally cultured cells. An in vivo study using rat models showed that the AFS had higher ability to perform the bone formation than commercially available, porous apatite ceramics. In this symposium, we will describe the development of the AFS with enhanced mechanical property and the results of in vivo evaluation using rabbit model as our recent works, together with the explanation of the current AF.

[1] M. Aizawa, H. Shinoda, H. Uchida, I. Okada, T. J. Fujimi, N. Kanazawa, H. Morisue, M. Matsumoto and Y. Toyama, Phosphorus Res. Bull., 17 262-268(2004).
[2] H. Morisue, M. Matsumoto, K. Chiba, H. Matsumoto, Y. Toyama, M. Aizawa, N. Kanzawa, T. J. Fujimi, H. Uchida, I. Okada, Spine, 31, 1194-2000(2006).
Enhancement of osteoconductivity and bioabsorbability of bone substitutes using partial dissolution-precipitation treatment

Yuichiro Abe

Department of Orthopaedic Surgery, Hokkaido University Graduate School of Medicine

Bone grafting is a commonly used procedure in orthopedic surgeries, and synthesized hydroxyapatite is mainly used as a bone substitute. Synthesized hydroxyapatite has a lower osteoconductivity and bioabsorbability compared to autologous bone graft, however, remains a serious problem. In this session, I show the favorable effect of partial dissolution-precipitation treatment on osteoconductivity and bioabsorbability of hydroxyapatite materials using animal models.		 Dentin as biomaterial- A case of immediate autograft of dentin and tooth -

Masaru Murata

Oral and Maxillofacial Surgery, School of Dentistry, Health Sciences University of Hokkaido.

Demineralized dentin matrix (DDM) is an osteoinductive, insoluble collagenous material. DDM granules derived from a vital tooth were absorbed and remodeled by new bone in tooth cavity revealing osteosclerosis.
The basic researches of human DDM and a clinical study of immediate autograft of DDM granules and a tooth, using our developed machines, will be introduced in this symposium.
Reconstruction of tissue-engineered bone using an apatite-fiber scaffold and a radial-flow bioreactor and its biological evaluation

Jun Fukasawa1), Yumiko Nakada1), Haruka Maehashi2), Tomokazu Matsuura2), and Mamoru Aizawa1)

1)Department of Applied Chemistry, School of Science and Technology, Meiji University
2)Division of Central Clinical Laboratory, Jikei University Hospital

We have successfully developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional (3D) inter-connected pores using single-crystal apatite fibers and carbon beads; subsequently, we have clarified that the AFSs have an excellent bioactivity on the basis of both in vitro and in vivo evaluations [1, 2]. In present study, in order to create the tissue-engineered bone with three-dimensional (3D) structure, we carried out 3D-cell culture of mesenchymal stem cells derived from rat bone marrow using the AFS settled into the radial-flow bioreactor. Some properties of the resulting tissue-engineered bone was examined through the histological and biochemical evaluations.
[1] M. Aizawa, H. Shinoda, H. Uchida, I. Okada, T. J. Fujimi, N. Kanazawa, H. Morisue, M. Matsumoto and Y. Toyama, Phosphorus Res. Bull., 17 262-268(2004).
[2] H. Morisue, M. Matsumoto, K. Chiba, H. Matsumoto, Y. Toyama, M. Aizawa, N. Kanzawa, T. J. Fujimi, H. Uchida, I. Okada, Spine, 31, 1194-2000(2006).		 Bone formation by porous chitosan /HAp nanocomposites with BMP-2 on rat parietal bone

Naoki Harada1), Haruhiko Kashiwazaki1), Tadashi Iizuka2 ), Makoto Demura3), Tomoyasu Aizawa3), Masaru Murata4), Junzo Tanaka5) and Nobuo Inoue1)

1) Department of Gerodontology,Hokkaido University
2) Support Section for Education and Research, Division of Oral Health Science, Graduate School of Dental Medicine,Hokkaido University
3)Faculty of Advanced Life Science, Hokkaido University,
Division of Oral and Maxillofacial Surgery,Hokkaido University
4)Department of Human Biology and Pathophysiology, Health Sciences University of Hokkaido ,
5)Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology

Porous chitosan /HAp nanocomposites with BMP-2 were implanted beneath the cranial periosteum of 4-week-old SD rats to assess bone development. At 4 weeks after implantation, the composites with BMP-2 resulted in active bone formation, whereas the composites alone showed no bone formation. These results suggested that the nanocomposite with BMP-2 was an effective biomaterial with osteoinductivity as an onlay graft substitute.
Interaction between proteins and carbon nanotubes: analyses by column chromatography and aggregation meter

Yoshinori Kuboki1), Hiroko Takita2), Ryuichi Fujisawa3), Takamitsu Koshikawa4), Shigeaki Abe4), Tsukasa Akasaka4), Motohiro Uo4), Fumio Watari4) Min-ho Lee 5),

1)Emeritus Professor, Hokkaido University
2)Support Section for Education and Research, Hokkaido University
3)Department of Oral Biochemistry and Molecular Biology,
Hokkaido University
4)Department of Biomedical Material and Engineering, Graduate School of Dental Medicine, Hokkaido University, Japan,Hokkaido University
5)Institute of oral Bioscience, Chonbuk National University, Korea

Type I collagen molecules and carbon nanotubes (CNT) share in their rod-like geometrical structures of nano-scale level. This similarity suggested us the unique interactions between both substances under certain circumstances. Actually, we previously discovered that strong aggregation occurred when native collagen was mixed CNT, but not the denature collagen (gelatin) iBio-Medical Materials and Engineering 19: 3-9, 2009). This phenomenon was further analyzed in the present study by applying chromatographic methods, leading many interesting findings.
 Clinical evaluation of masticatory restration with dental implant for total edentulous maxilla and mandible.

Eizaburo Kobayashi1),Kazuhisa Mori1),Izumi Mataga1),Fumihiko Watanabe2)

1)Department of Oral and Maxillofacial Surgery, School of Life Dentistry at Niigata,The Nippon Dental University
2)Department of Crown and Bridge, School of Life Dentistry at Niigata,The Nippon Dental University

In recent, masticatory function become better by improvement of stability of proshtetic devices placed on the dental implant. In this report, clinical evaluation for these patients who were treated in our department by prosthetic reconstruction using dental implant were retrospectively performed.
Thirty-one total edentulous patients from total number of all 307 patients who were installed dental implant in the Niigata Hospital, School of Life Dentistry at Niigata, The Nippon Dental University for 19 years from 1989 through 2008 were respectively discussed.
In these 31 patients, 15 were males and 16 were females, aged from 42 to 70 years old, average of 60.7. According to a location installed implants, maxilla was 12 and mandible was 23. Both edentulous maxilla and mandible was 4 patients.
Clinical study of sinus floor augmentation with b-tricalcium phosphate

Hiroyuki Kitajo, Takeo Maita, Hiroki Nagayasu, Junichi Tazaki1), Masaru Murata2),Makoto Arisue2), Takanori Shibata1)

1)Institute of personalized medical Science, Oral and Maxillofacial Surgery, Health Sciences University of Hokkaido
2)Reconstructive Surgery for Oral and Maxillofacial Region, Health Sciences University of Hokkaido

Maxillary sinus augumentation is a method of attaining sufficient bone height@for posterior maxillary implant placement. The excellent biocompatibility of calcium phosphate ceramics has lead to their extensive use as biomedical materials. Beta-tricalcium phosphate (ƒÀ-TCP) appears to be advantageous in comparison to HAp for the property of absorption with bone remodeling. In this study, 52-year-old femalepresented with atrophied upper jaw was performed sinus floor augmentation with@ƒÀ-TCP and Platelet Rich Plasma (PRP). At 6 months after implantation, the place of bone augmentation was trephined and investigated clinically and pathologically.
Bisphosphonate-related Osteonecrosis of the Jaw - case report -

Yu-Feng Huang1), Jie Yang2), Ming-Yung Chou1)

1)Oral Medicine Center, Chung Shan Medical University Hospital, Taichung, Taiwan
2)Oral maxillofacial Radiology, School of Dentistry, Temple University, Philadelphia, PA, USA

Bisphosphonates were developed in the 19th century, but were first investigated in the 1960s for use in disorders of bone metabolism. The initial rationale for their use in humans was their potential in preventing the dissolution of hydroxylapatite, the principal bone mineral, and hence arresting bone loss by inducing apoptosis in osteoclasts and monocytic precursor cells. After decades of use, many side effects have been reported including osteonecrosis of the jaw. Here we reported a case of bisphosphonate-related osteonecrosis which we analyzed with cone-beam CT. Patient is a 76 y/o male who was diagnosed as prostate cancer 2 years ago with bone metastasis. He has been receiving ZometaR injection once a month for 12 months. He had had #43, #44 extracted at local dental clinic due to severe mobility one month ago. A few days later, he developed painful swelling at right mandibular body region. At the time of examination, there was only #34 retained in the mouth. Significant swelling of right mandible was noted with abscess draining from the mandible. A bony hard swelling was observed at right inferior border of chin. Cone-beam CT was performed and the images were analyzed with Cybermedfs Ondemand 3D. All features are consistent with bisphosphonate-related osteonecrosis with secondary infection. Continuous antibiotic was provided for 10 days and pus discharge was reduced significantly and not painful, but the swelling of mandible remained. Due to the wide extent of the osteonecrosis and patientfs status, surgical debridement was not recommended. Patient is still on weekly follow up.
Proteins in enamel lamella of porcine mature enamel

Rumi Nakamura1), Ryuji Yamamoto2), Makoto Fukae2), Yasuko Momoi1)

1)Department of Operative Dentistry, Tsurumi University School of Dental Medicine
2)Department of Biochemistry, Tsurumi University School of Dental Medicine

The purpose of this study is to investigate the components and formation processes of enamel lamella of porcine mature enamel after demineralization with 0.5M acetic acid. SDS electrophoresis, Mass spectrometry and Western blotting analysis showed that the structure contained a lot of proteins, in which the blood derived proteins such as albumin, hemoglobin and their degradation products were mainly resided. Since the degradation products were observed in the low-molecular area after electrophoresis, it was considered that the blood derived proteins were not contamination of the blood during sample preparation, but were original component of the products. In addition of them, there were a small amount of enamel proteins, collagen and DSPP.		 Histological study of new CaTiO3-aC coating material ?in vivo study-

Ryo Tamamura1), Miho Inoue1), Yasuhisa Hirata1),2), Midori Kubota1), Shin Takagi2), Etsuo Kishimoto3), Tohru Takagi1), Masahisa Inoue4), Hitoshi Nagatsuka1) and Noriyuki Nagai1)

1)Department of Oral Pathology and Medicine, Tokushima Bunri University
2)Department of Oral and Maxillofacial Reconstructive Surgery,Tokushima Bunri University
3)Department of Oral Health, Tokushima Bunri University
4)Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Laboratory for Structure and Function, Faculty of Pharmaceutical Sciences, Tokushima Bunri University

In this study, the objective was to evaluate the effect of CaTiO3-aC coating material on bone tissue by in vivo study. Titanium bars with CaTiO3-aC coating or HA coating or without coating as a control were implanted in rat tibias. After 1 and 2 weeks post implantation, implants were removed with surrounding bone tissue and histological examination was performed. In the experimental group, plenty of new bone formation around titanium were observed as compared with control. Especially, in CaTiO3-aC coating group, the implant was in contact with new bone. Our results suggest that CaTiO3-aC plays a dominant effect on tissue response, which could be considered as an important biomaterial for dental implants.
The influences of Colloidal Platinum Nanoparticles on resin/ root dentin adhesion using 4-META/MMA-TBB resin.

Futami Nagano1), Kazuhiko Endo1), Masanori Hashimoto1), Hidehiko Sano2), Yusei Miyamoto3), Hiroki Ohno1)

1)Biomaterials and Bioengineering, Health Sciences University of Hokkaido
2)Restorative Dentistry, Hokkaido University
3) Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo

Our previous studies showed that pre-treatments of coronal dentin surface with Colloidal Platinum Nanoparticles (CPN) significantly improved the bond strength between SuperBond C&B (4-META/MMA-TBB) and coronal dentin. The aim of this study is to evaluate the influence of the pre-treatment with CPN on resin/ root dentin adhesion using SuperBond C&B. The pre-treatments were performed as following. Control: Etching with 10-3 solution. Etch-CPN: Etching then CPN application. CPN-Etch: CPN application then etching. After PMMA blocks were adhered by SuperBond C&B, specimens were kept in 37Ž water for 24 h and they were exposed to the micro tensile bond strength test. The results of the bond strength showed no significant difference between 3 groups.		 Nicotine and LPS increase MMPs and tPA production and decrease TIMPs production in osteoblasts

Tomoko Katono1), Takayuki Kawato1),3), Natsuko Tanabe1), 3), Hideki Tanaka*,Takafumi Iida1), Masafumi Motohashi1), 3), Naoto Suzuki2),3), Kichibee Otsuka2),3), Masao Maeno1),3)

1)Departments of Oral Health Sciences,Nihon University School of Dentistry
2)Biochemistry, Nihon University School of Dentistry, Nihon University School of Dentistry
3Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry

The arm of this study is to investigate the effect of nicotine and/or LPS on turnover in osteoid by osteoblasts. Nicotine and/or LPS increased MMP-1, -2, and -3 and tPA production; decreased TIMP-1, -3, and -4 production. In the presence of D-tubocurarine or polymyxin B, neither nicotine nor LPS stimulated MMP-1 production. In the presence of NS398 or celecoxib, the stimulatory effects of nicotine and LPS on MMP-1 production were unchanged, but they were unable to stimulate PGE2 production.@These results suggest that nicotine and LPS stimulate the resorption process that occurs during turnover of osteoid. Furthermore, they suggest that the stimulatory effect of nicotine and LPS on PGE2 production is independent of their stimulatory effect on MMP-1 production.
Mechanical stress induces osteoclast differentiation via PGE2 production in osteoblasts

Rina Sanuki1), Narihiro Mitsui1), Masao Maeno2), 3), Naoto Suzuki3), 4), Kichibee Otsuka3), 4), Hideki Tanaka2), Yuki Koyama1), Chieko Shionome1), Fan Zhang6), Noriyoshi Shimizu, 1),5)

1)Departments of Orthodontics, Nihon University School of Dentistry
2)Oral Health Sciences,Nihon University School of Dentistry
3)Biochemistry, Nihon University School of Dentistry
4)Divisions of Functional Morphology,Nihon University School of Dentistry
5)Clinical Research, Nihon University School of Dentistry
6)Dental Research Center, Nihon University School of Dentistry and Departments of Orthodontics, Shandong University School of Dentistry

The present study was undertaken to examine the effect of mechanical stress (MS) on PGE2, COX-2, M-CSF, RANKL, and OPG production in osteoblasts, and to examine the indirect effect of MS on osteoclast differentiation. Osteoclast differentiation was estimated using TRAP staining of osteoclast precursors cultured with conditioned medium from MS-treated osteoblasts. As MS increased, PGE2, COX-2, M-CSF, and RANKL production increased, whereas OPG production decreased. The number of TRAP-positive cells increased as MS increased. Celecoxib blocked the stimulatory effect of MS. These results suggest that MS induces osteoclast differentiation by increasing M-CSF and RANKL production and decreasing OPG production via PGE2 in osteoblasts.		 Establishment of odontoblastic stem cells producing extracellular matrix under in vitro condition

Hidetsugu Tsujigiwa1), Naoki Katase2), Eiki Yamachika3), Hitoshi Nagatsuka2), Andrea P. Rodriguez2), Hidemitsu Harada4), Masao Yamada1)

1)Department of Virology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
2)Department of Oral Pathology and Medicine,Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
3)Department of Oral and Maxillofacial Reconstructive Surgery,Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
4)Department of Oral Anatomy II, School of Dentistry, Iwate Medical University

We have established odontoblastic stem cells (OSC) from the dental pulp cells of GFP transgenic rats. OSC showed increased ALP activity, production of dentin related proteins and positive reaction for alizarin red staining, and produced extracellular matrix when cultivated in the presence of b-GP. Co-culture of OSC and ameloblastic stem cells (ASC) demonstrated that OSC facilitated calcification of ASC. Moreover, OSC formed dentin-like calcified tissue in vivo when transplanted into dorsal subcutaneous tissue of immune-deficient mice. All the results suggested that this established OSC possessed same biological characters as OSC in vivo, and thought to be useful for tooth regeneration researches and elucidation of cell differentiation mechanism in odontogenesis.
IL -1b suppresses the formation of osteoclasts by increasing OPG production via an autocrine mechanism involving celecoxib-related PGs in chondrocytes

Yusuke Watanabe1), Yukiko Aida1, 4), Masao Maeno3),4) , Nakayama Go1),Naoto Suzuki2), 4), Kichibee Otsuka2), 5), Hideo Matsumura1), 4)

1)Departments of Fixed Prosthodontics, Nihon University School of Dentistry
2)Biochemistry, Nihon University School of Dentistry
3)Oral Health Sciences, Nihon University School of Dentistry
4)Divisions of Advenced Dental Treatment, Nihon University School of Dentistry
5)Functional Morphology, Dental Research Center, Nihon University School of Dentistry

We examined the effect of IL-1ƒÀ and/or celecoxib on the expression of M-CSF, RANKL, and OPG in human chondrocytes, and the indirect effect of IL-1ƒÀ on osteoclast-like cell formation using RAW264.7 cells. OPG and RANKL expression increased with IL-1ƒÀ, whereas M-CSF expression decreased. Celecoxib blocked the stimulatory effect of IL-1ƒÀ on OPG and RANKL expression. Conditioned medium from IL-1ƒÀ-treated chondrocytes decreased TRAP staining. These results suggest that IL-1ƒÀ suppresses the formation of osteoclast-like cells via increased OPG production and decreased M-CSF production in chondrocytes, and OPG production may increase through an autocrine mechanism involving celecoxib-related PGs.		 IL-17 suppresses the expression of proteases and differentiation in osteoclast precursors

Satoshi Kitami1), Hideki Tanaka1), Takayuki Kawato1),3), Natsuko Tanabe1),3), Akiko Kuwabara1), Toyoko Morita1), Masafumi Motohashi1),3), Naoto Suzuki2),3), Kichibee Otsuka2), 3), Masao Maeno1), 3)

1)Departments of Oral Health Sciences, Nihon University School of Dentistry
2)Biochemistry, Nihon University School of DentistryHealth Sciences University of Hokkaido
3)Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry

The arm of this study is to investigate the effect of IL-17 on osteoclast differentiation and the expression of carbonic anhydrase II (CA2), MMP-9, and cathepsin K (CK) using RAW264.7 cells as osteoclast precursors. RAW264.7 cells were cultured with or without IL-17 in the presence of soluble RANKL for up to 10 days. TRAP-positive cells were detected on day 5 of culture, and the number of the cells decreased by the addition of IL-17. The gene expression of CA2, MMP-9 and CK in the cells also was decreased by the addition of IL-17 on day 5 of culture. These results suggest that IL-17 suppresses osteoclast differentiation and the function by decreasing of CA2, MMP-9, and CK.
Mesenchymal stem cell-like potential of periodontal ligament fibroblasts to construct vascular cell-specific marker-positive blood vessel structure with a lumen

Naoto Okubo1),2),3), Masato Tamura2), Tadashi Iizuka4), Masaharu Kamo1), Seiko Kyakumoto1), Naoyuki Chosa1), Noriko Takahashi1), Yoshimasa Kitagawa2), and Akira Ishisaki1)

1)Department of Biochemistry, Iwate Medical University, School of Dentistry, Hokkaido University
2)Department of Oral Biochemistry and Molecular Biology, Division of Oral Health Science,Hokkaido University
3)Department of Oral Diagnosis and Oral Medicine, Division of Oral Pathobiological Science, Hokkaido University
4)Support Section for Education and Research, Graduate School of Dental Medicine, Hokkaido University

Objective: To evaluate whether fibroblasts derived from periodontal ligament (PDL) retain the ability to differentiate into putative vascular cells and construct blood vessel structures.
Methods: Single cell-derived cultures (SCDCs) were established from primary rat PDL fibroblast cultures, and their expression of mesenchymal stem cell (MSC)- and endothelial cell (EC)-markers was evaluated by RT-PCR. The ability to construct a blood vessel structure was evaluated in three-dimensional cultures. The morphological and immunohistological characteristics of the structure were then evaluated.
Results: Each SCDC expressed EC in addition to MSC markers. SCDC2 cells, which abundantly expressed Tie-2, vigorously constructed a blood vessel structure.
Conclusion: PDL fibroblasts have the potential to construct an EC marker-positive blood vessel-like structure.		 Tissue regeneration using rhPDGF-BB with gelatin sponge

Atsushi Niida1), Yukito Hirose1), Morio Ochi1), Masaru Murata2), Tohru Kaku3)

1)Division of Fixed Prosthodontics and Oral Implantology, Department of Oral Rehabilitation, Health Sciences University of Hokkaido
2)Division of Oral and Maxillofacial Surgery, Health Sciences University of Hokkaido
3)Division of Clinical Oral Pathology, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido

After bone augmentation using biomaterials, the failure of the suture sometimes occurs. The aim of this study is to achieve the conglutinant suture part, because there are troubles on the microcirculation and tension between tissue of suture part and mucosa membrane. Three by 4 mm in size of cross-linked gelatin sponges were inserted into Teflon tube. After that, 10?ng of diluted rhPDGF-BB was dripped on them and implanted into the subcutaneous tissue of 8-month-male ratsf back. After 2 weeks, the materials were explanted and microscopically observed. In the PDGF group, the fibrous tissue invaded into sponge, while the tissue invasion in the sponge alone group was not seen. In these results, PDGF sponges may be useful for the tissue regeneration.
Enhancement by the peptide hydrogel of osteogenesis induced by rhBMP-2 in rats

Hiroko Takita1), Tadashi Iizuka1), Yoshinori Kuboki2)

1)Support Section for Education and Research, Graduate ,Hokkaido University
2)School of Dental Medicine, Hokkaido University
Hokkaido University

The aim of this study is to investigate the effect of the peptide hydrogel (PH) on BMP-induced ectopic bone formation. The peptide hydrogel can compose optimal 3D spaces. A fibrous glass membrane as a BMP carrier was mixed with PH and BMP-2. The mixed composites were implanted into rats subcutaneously. The results showed that Ca and osteocalcin contents of PH/BMP implants were higher than those of BMP implants at 3 weeks after implantation. It is concluded that the BMP-induced bone formation depends upon the geometry of the carrier, which can be constructed with PH.		 A case of calcifying epithelioma in cheek region with osteocyte and bone formation - hard tissue-related genetic analysis-

Kaoru Kusano1), Katsutoshi Ito1), Jun Hino1), Masaru Murata1), Hiroki Nagayasu2) , Yoshihiro Abiko2) , Tohru Kaku3) Takanori Shibata4) and Makoto Arisue1)

1) Oral and Maxillofacial Surgery, Health Sciences University of Hokkaido
2) Institute of Personalized Medical Science, Health Sciences University of Hokkaido
3 Clinical Oral Pathology, Health Sciences University of Hokkaido
4 Reconstructive Surgery for Oral and Maxillofacial Region, Health Sciences University of Hokkaido

Calcifying epithelioma is a benign tumor derived from hair-matrix cells, a rare lesion in the oral and maxillofacial regions. The patient was a 33 years old female, who was aware of a consistent painless tumor in the right cheek for 20 years. The tumor was of bone-like hardness and immobile under the skin with the size of the tip of the little finger. The clinical diagnosis was calcifying epithelioma, and tumorectomy was performed under local anesthesia and intravenous sedation. The histopathological findings of the tumor revealed shadow cells with osteocytes and bone formation. Based on clinical and histopathological findings, the patient was diagnosed with a calcifying epithelioma. Total RNA was extracted from the removed tumor. Hard tissue-related gene expressions(BMP-2CALPCOPNCOCN)were determined by RT-PCR, and hard tissue-related mRNA expressions were observed in the removed tissue. These results indicated that hard tissue-related mRNA is associ
Application of the adipose tissue-derived stem cell to the bone tissue engineering

Masanori Takekawa, Mituyoshi Matsuda, Eiji Kondoh and Hiroya Ito

Department of Oral and Maxillofacial Surgery, Asahikawa Medical College

The aim of this study is to assess the usefulness of adipose tissue-derived stem cells(ADSCs) for bone tissue engineering. Collagen sponges(CS) with ADSCs were cultured in osteogenic differentiation medium. At the 2nd week of the culture, the samples were implanted to ratfs subcutaneous tissues. 4 weeks after implantation, samples were examined. For the control, cells were cultured without osteogenic differentiation medium. A large quantity of newly formed bone was observed in the inside of CS. In the control, new bone formations were not observed. The results suggested that the adipose tissue-derived stem cell was useful for bone regenerative medicine.		 Effects of IL-6 and sIL-6R on turnover of bone and cartilage matrix proteins in chondrocytes

Kazuhiro Honda1), Yukiko Aida1),3), Masao Maeno2), 5), Yusuke Watanabe1), Shihoko Tanigawa1), Suzuki Naoto3),5), Kichibee Otsuka3), 5), Hideo Matsumura1), 4)

1)Departments of Fixed Prosthodontics, Nihon University School of Dentistry
2)Oral Health Sciences,Nihon University School of Dentistry
3)Biochemistry, Nihon University School of Dentistry
4)Nihon University School of Dentistry, Divisions of Advanced Dental Treatment, Nihon University School of Dentistry
5)Functional Morphology, Dental Research Center, Nihon University School of Dentistry

We examined the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on the expression of MMPs, PAs and their inhibitors (TIMPs and PAI-1) in human chondrocytes. By the addition of IL-6 and sIL-6R, the expression of MMP-1, -3, -13 and TIMP-1 increased for the culture period of 28 days; the expression of MMP-1, -13 and TIMP-1 increased markedly. The expression of TIMP-2, -3 and -4 increased after days 7 or 14 of culture. On the other hand, MMP-2 and uPA expression decreased for the culture period of 28 days and tPA expression decreased after day 14 of culture. These results suggest that IL-6 and sIL-6R stimulate turnover of bone and cartilage matrix proteins by increasing collagenases and TIMP-1 in chondrocytes, and also that plasminogen/plasmin systems in the cells are not related in the turnover.
Compressive force stimulates the expression of IL-17s and their receptors in osteoblastic MC3T3-E1 cells

Fan Zhang1), Yuki Koyama1, Masao Maeno2),4), Narihiro Mitsui1), Chieko Shininime1), Kotoe Mayahara1), 5), Naoto Suzuki3),4), Kichibee Otsuka3),4), Noriyoshi Shimizu1), 5)

1)Departments of Orthodontics,Nihon University School of Dentistry
2)Oral Health Sciences,Nihon University School of Dentistry
3)Biochemistry, Nihon University School of Dentistry
4)Nihon University School of Dentistry, Divisions of Functional Morphology,Nihon University School of Dentistry
5)Clinical Research, Dental Research Center, Nihon University School of Dentistry

Recently, interleukin (IL)-17 was found to stimulate osteoclastic resorption via osteoblasts by inducing RANKL expression. We examined the effect of compressive force on the gene expressions of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, and their receptors (IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE) using MC3T3-E1 cells as osteoblasts. The expression levels of IL-17s and their receptors increased depending on the compressive force. These results indicate that mechanical stress induces the expression of IL-17s and their receptors in osteoblasts, and that IL-17s and their receptors produced in response to mechanical stress may affect osteoclastgenesis via RANKL expression.		 Radiographic Demonstration of the Soft Tissue Using the New Type of Source of X-ray PXR

Yasuko Numata1),Toshiro Sakae1), Hirotsugu Yamamoto1), Takasi Kaneda1), Jin Yamamoto1), Isamu Sato2),Yasushi Hayakawa3),Yumiko Takahashi3), Kyoko Nogami3)

1)Nihon University School of Dentistry at Matsudo,
2)Nihon University Graduated School,
3)Laboratory for Electron Beam Research and Application of Nihon University.

This study aimed to take X-ray photographs of soft tissue, which was difficult to do with conventional X-rays, using a new X-ray source of PXR (Parametric X-ray) generated in LEBRA. At first the hard tissue was photographed by direct imaging and was able to get clear radiograph, showing the PXR has ability for such purpose. Applying a phase contrast imaging for soft tissues and/or materials composed of light elements using the PXR resulted in good appearance of images, whereas it is difficult to do so by a conventional white X-rays. In future, developing these characteristics of LEBRA-PXR may introduce a great improvement in diagnosis and treatment in the medical scene. (Supported in part by Nihon University Multidisciplinary Research Grant for Sogo 09-024, 2009)
Elemental Analysis of Arsenic Using a Wavelength-Tunable Parametric X-ray

T. Sakae1), T. Hayakawa1), K. Suzuki1), Y. Tanimoto1), H. Nakada1), I. Sato2), T. Shintomi2),H. Yamamoto3), K. Suzuki3), N. Iwata3), T. Tanaka4), K. Hayakawa4), Y. Hayakawa4), Y. Takahashi4), T. Kuwada4)

1)Nihon Univ. Sch. Dent. at Matsudo,
2)Nihon Univ. ARISH,
3)Nihon Univ. College of Science and Technology,
4)Nihon Univ. LEBRA,

Arsenic is known as not only valuable element used in GaAs semiconductor, but as harmful for human body. Tolerance of human intake is 0.05 mg/kg/day. Analysis of As is usually carried out using Atomic Absorption Analysis or LC-ICP/MS, but these techniques do not allow in vivo analysis. Using a wavelength-tunable Parametric X-ray generated at LEBRA, Nihon University, the X-ray absorption edge for As in realgar was clearly detected. Applying this PXR for radiography may be able to detect and quantify the harmful heavy metals in human body. (Supported by Nihon University Multidisciplinary Research Grant for@Sogo09-024, 2009)		 Studies on the distribution of acellular and cellular cementum in human permanent teeth

Hitoshi Yamamoto, Toshihide Niimi, Rumi Yokota-Ohta, Kunihiro Suzuki, Toshiro Sakae, Yukishige Kozawa

Department of Histology, Cytology and Developmental Anatomy,
Nihon University School of Dentistry at Matsudo

Cementum is divided into acellular and cellular cementum. It is generally known that acellular cementum is located on the cervical portion and cellular cementum distributes both on the apical portion and furcation area. In fact, however, acellular and cellular cementum are distributed irregularly. The authors observed grinding sections of extracted human teeth (50 incisors and 78 molars) by light microscopy. As a result, the distribution of acellular and cellular cementum showed a variety of patterns as well as the typical distribution pattern.
HSP27 expression in malignant transformation of ameloblastoma

Keisuke Nakano1), Chong Huat Siar2) and Toshiyuki Kawakami1)

1)Hard Tissue Pathology Unit, Matsumoto Dental University Graduate School of Oral Medicine,
2)Department of Oral Pathology, Faculty of Dentistry, University of Malaya

Heat shock protein (HSP) expression caused by various kinds of stress and HSP is responsible for cytological regulation of cell differentiation, proliferation and/or morphogenesis. In this research, we examined HSP27 peptide in both ameloblastoma and ameloblastic carcinoma. After histopathological examination, the distribution of HSP27 was examined by immunohistochemistry (IHC) using anti-human HSP27 monoclonal antibody. Ameloblastoma showed strong HSP27 reactivity in columner and polyhedral tumor cells. In the other hand, ameloblastic carcinoma showed HSP27 reactivity in atypical tumor cells. These results suggest that HSP27 plays some role in cytological differentiation and progression of malignant transformation in ameloblastoma.		 Expression of heparanase in odontogenic cystic lesions

Hiroyuki Matsuda1), Naoki Katase1), Hidetsugu Tsujigiwa2), Gul San Ara Sathi1), Masae Fujii1), Mathieu Lefeuvre1), Han Liu3), Ryo Tamamura1) and Hitoshi Nagatsuka1)

1)Department of Oral Pathology and Medicine ,, Okayama University
2)Department of Virology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University,
3)Department of Oral Biology, College of Stomatology, Dalian Medical University

The aim of this study was to clarify the expressions of heparanase protein and gene in odontogenic cystic lesions. Heparanase expressions in follicular cysts (FC), radicular cysts (RC), keratocystic odontogenic tumors (KCOT) and ameloblastomas (unicystic type, A-U) were examined by immunohistochemistry and in situ hybridization. In all cystic lesions except RC, the heparanase expressions were observed in epithelial component. Intense gene expression was observed in KCOT associated with naevoid basal cell carcinoma syndrome compared with sporadic ones. These results suggest that heparanase may play a role of tumor growth
Effect of oral administration of statin on bone regeneration process in ovariectomized rat

Hata Saya1), Tohru Hayakawa2), Makiko Ono1), Hirotsugu Yamamoto3), Yoshiaki Akimoto1)

1)Department of Oral Surgery,Nihon Univeresity School of Dentistry at Matsudo
2)Department of Dental Biomaterials,Nihon Univeresity School of Dentistry at Matsudo
3)Department of Oral Pathology, Nihon Univeresity School of Dentistry at Matsudo

It has been reported that simvastatin (SV), which is a cholesterol synthesis inhibitor and a therapeutic drug for hypercholesteremia. The purpose of this study is to evaluate the efficacy of oral administration of SV on the bone healing process in the cortical bone defects of the tibiae of ovariectomized (OVX) rats. In conclusion, the present results revealed that oral administration of SV might provide a beneficial effect on bone healing process for OVX rats.