Research Projects -
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自己骨髄由来間葉系幹細胞にタンパク質導入法を応用した歯槽骨再生技術の開発
Grant number：16659534 2004 - 2005
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
完山 学, 窪木 拓男, 荒川 光, 縄稚 久美子, 小島 俊司
Grant amount：\3200000 （ Direct expense: \3200000 ）
本研究は,タンパク質導入法を用いて,BMP,SHH,FGF,IGF,TGF-b,CTGFなど骨形成に関連する成長因子を自己骨髄由来間葉系幹細胞内に導入し,次にそれらをキャリアとともに抜歯窩や実験的な歯槽骨欠損部位に移植することで歯槽骨の再生を試みようとするものである。
昨年度は,FITCとCTGF,そのファミリーであるcyr61,novなどの骨形成関連遺伝子のクローニングを行った。今年度はBMP-2,-4,SHH,FGF-2,galectin-1,-3,-9,sod-1など骨形成に関連する成長因子をコードするcDNAのクローニングを行った。現在,これらの遺伝子にPTD配列を含んだ遺伝子を増幅しPETベクターにクローニングを行っている。
in vivo実験においては,昨年度行った抜歯窩モデルが実験群とコントロール群で差が認められなかったことから今年度は大きな骨欠損モデルで試みた。はじめに生後8週齢のWistar系雄性ラット頭蓋骨全層骨欠損モデル(critical size defect)を作製した。これはラット頭蓋骨に直径6mmの全層骨欠損を作成したもので,この欠損部にBMP-2,FGF-2を含浸させた直径6mmのゼラチンハイドロゲルシート,I型コラーゲンシートを移植したところ,術後4週でゼラチンハイドロゲルシートもしくはI型コラーゲンシートとBMP-2を用いたものは,BMP-2単体やbFGF単体と比較して著名な骨再生が認められた。しかし,再生部位を歯槽骨に近づけるべく3.0kg雄性日本白色ウサギ下顎骨骨欠損モデル(defect size : 6mm)を用いた場合,I型コラーゲンキャリアとbFGFを用いても術後4週でコントロールとの著名な差を見いだすことができなかった。
今後は,PTD配列を含んだ骨形成関連遺伝子とゼラチンハイドロゲルやI型コラーゲンキャリア,さらに骨髄由来間葉系幹細胞を組み合わせた実験系を構築する予定である。 -
Development of functional Ti implants with release of growth factors
Grant number：16390557 2004 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
YOSHIDA Yasuhiro, SUZUKI Kazuomi, KUBOKI Takuo, HIRATA Isao, TAGAWA Yoh-ichi, NAGAOKA Noriyuki
Grant amount：\13800000 （ Direct expense: \13800000 ）
In order to develop functional Ti implants with release of growth factors, biodegradable materials as a carrier have to adhere strongly to Ti surface. Lactide copolymers synthesized in this study hardly adhered to TI surface.
Ti plates were treated with 0.1 and 1 N hydrochloric acid, 37wt% phosphoric acid or kept untreated. The tensile bond strength of the composite cement to the untreated and pre-treated Ti plates was determined without and after 20,000 thermo-cycles. XPS was used to chemically analyze the effect of the three Ti pre-treatments, as well as the interaction of 10-methacryloxydecyl dihydrogen phosphate (10-MDP) with Ti treated with 1 N HCl. Although no significant difference in immediate tensile bond strength was measured, thermo-cycling significantly decreased the bond strength of all experimental groups except for Ti treated with 1 N HCl. XPS demonstrated that H_3PO_4 was strongly adsorbed on the Ti surface. These results clearly suggest that phosphorylation was effective to get strong attachment to the Ti surface. Ti pre-treated with 1 N HCl improved the adsorption of 10-MDP as compared to untreated Ti.
Cleaned Ti disks were immersed into three different concentrations of polyphosphoric acid solution (0.1,1 and 10 wt%) for 24 hours at 37℃. Ti immersed in distilled water for 24 hours at 37℃ served as control. Degrees of cell attachment (1,3,5 hours after cell seed) and proliferation (1,3,5 and 7 days after cell seed) on each treated Ti disk were evaluated by MTS assay. A significantly higher cell attachment was found on Ti treated with polyphosphoric acid in contrast to untreated Ti (control) for all three culture periods. MTS assay also revealed that cell proliferation levels significantly increased following a polyphosphoric-acid dose dependency. It was concluded that polyphosphoric-acid treatment of Ti enhanced the attachment and proliferation of hBMSCs. -
Establishment of an autologous cell transplantion method using mesenchymal stem cells for alveolus bone regeneration.
Grant number：16591947 2004 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
FUJISAWA Takuo, KUBOKI Takuo, TAKIGAWA Masaharu, UEHARA Junji
Grant amount：\3100000 （ Direct expense: \3100000 ）
The purposes of this research are to investigate the possibility of autologous mesenchymal stem cell transplantion method for alveolus bone regeneration using connective tissue growth factor (CTGF).
1. Isolation of human bone marrow cells and cell characterization.
Bone marrow cells were isolated from human iliac bone marrow of volunteer. Human bone marrow cells were plated and cultured in DMEM containing 10% FBS. Moreover, STRO-1 expressing cells were identified by immunostaining from culture of passage 9 cells, and these cells showed the multilineage differentiation (osteoblastic and adipogenic).
2. Effects of CTGF on stem cells behavior.
1) Cell attachment efficiency onto hydroxyapatite disks is enhanced by coating CCN2/CTGF dose-dependently, and CCN2/CTGF activated p38 MAPK signal pathway via αvβ3 integrin.
2) CCN2/CTGF significantly increased the cell proliferation dose dependently.
3) The migration assay using Chemotaxicell indicated that CCN2/CTGF significantly increased the cell migration.
4) CCN2/CTGF did not affect cell differentiation to the osteoblast.
5) CCN2/CTGF also enhanced the endothelial cell attachment and proliferation.
6) In vivo implantation study indicated that CCN2/CTGF enhance the stem cell survival and induce their migration into the porous hydroxyapatite scaffold. -
Cloning, Expression and Identification of Specific Genes related Osseointegration to Titanium.
Grant number：15390592 2003 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
KANYAMA Manabu, KUBOKI Takuo, TAKIGAWA Masaharu, ARAKAWA Hikaru, SUZUKI Kouji, FUJISAWA Takuo
Grant amount：\14800000 （ Direct expense: \14800000 ）
The aim of this study is to clarify the cell behavior induced by titanium and to isolate specific genes that promote the titanium implant-bone integration. Especially, to identify the master key genes related osseointegration to titanium, the osteoblastic cell line, MC3T3-E1 cells were cultured on titanium coated glasses and the gene expression were analyzed.
1)Titanium and chrome coated glasses
Ti and Cr were coated to polished glass surfaces (33 mm in diameter by 1.5 mm in thickness) in a high vacuum condition. Ti and Cr coated glass disks were placed in a 6 well plate and fixed by silicone rings. Non-coated glass disks were served as controls.
2)Cell attachment, proliferation and differentiation assessments.
Initial cell attachment and proliferation of MC3T3-E1 cells were estimated by MTS-assay (CellTiter 96【○!R】 AQueous One Solution, Promega, USA), and cell differentiation was evaluated by alkaline phosphatase activity assay. The mean relative amount of the attached cells on the Ti-coated glass was significantly higher than those on non-coated and Cr-coated glass at 3 hours after seeding. On the same way the mean cell number on Ti-coated glass at 3 days after seeding was significantly higher than those of other conditions. The mean alkaline phosphatase(ALP) activity of the seeded osteoblasts also increased with time in these three conditions, while the ALP activity level on the Ti-coated glass at 14 days after seeding was significantly higher than those of other conditions. These results suggest that, Ti-coated glass plate accelerated the cell attachment, proliferation and differentiation of the MC3T3-E1 cells, compared to the non- and Cr-coated glass plates
3)Effect of gene expression of osteoblast on titanium
Osteoblasts were cultured on Ti coated, Cr coated and non-coated glass plates. And then differentially expressed genes were identified by cDNA subtractive hybridization. Twenty independent clones were isolated and by nucleotide sequencing of these clones, seven clones were identified including EST genes ; xab-2,sod-1,galectin-1,actin related protein 2/3 mRNA, RIKEN cDNA 2210013021 gene, EST 601086505F1, and EST 01439. And galectin-1,xab-2,sod-1 gene expression on Ti-coated glass were higher than non-coated, Cr-coated glass.
These results suggest that Ti-coating is more advantageous for osteoblastic cell attachment, proliferation and differentiation than Cr or non-coating and galectin-1,xab-2 and sod-1 up-regulation could be related to the Ti-bone integration. -
The trial of treatment for temporomandibular joint osteoarthritis with soluble tumor necrosis factor receptor
Grant number：15592050 2003 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MAEKAWA Kenji, FUJISAWA Takuo, KUBOKI Takuo, UEHARA Junji
Grant amount：\3400000 （ Direct expense: \3400000 ）
In this study, we tried to confirm soluble tumor necrosis factor receptor(sTNFR)-I,II concentrations in synovial fluids of osteoarthritic temporomandibular joint (OA) and to investigate the expression changes of sTNFR-I,II in the articular chondrocytes under an osteoarthritis-mimicking condition.
1.sTNFR-I and -II concentrations were higher in the synovial fluids from OA patients than from asymptomatic controls.
2.sTNFR-I concentration was higher than sTNFR-II concentration in the synovial fluids from both of patients and asymptomatic controls.
3.sTNFR-I concentration in the synovial fluids was significantly related to the OA level.
4.Higher sTNFR-II concentration in the synovial fluids was associated with less pain and less restricted range of mouth opening in the osteoarthritic patients.
5.In the knee osteoarthritis rat model, both of TNFR-I and TNFR-II were found at the knee joint chondrocyte of arthritic side and control side, and in the arthritic side both of receptors were up-regulated.
6.IL-1β and/or TNFα addition to the culture medium up-regulated tnfr-I,-II gene expression levels of the cultured chondrocytes and specifically sTNFR-II protein concentration in the cultured medium.
Since IL-1β and/or TNFα addition to the culture medium up-regulated tnfr-II gene expression levels of the cultured chondrocytes and sTNFR-II protein concentration in the cultured medium, observed sTNFR-II up-regulation in the synovial fluids of the osteoarthritic patients could reflect the protective chondrocyte metabolism of the osteoarthritic joint. Even in the osteoarthritic joints, sTNFR-II could modulate inflammation and destruction of the temporomandibular articular tissues.
Therefore, novel therapies may include agents that specifically mimic the anti-inflammatory feedback mechanism of sTNFR-II in vivo. We speculate that injection into temporomandibular joint with Etanercept, a potent inhibitor of TNFα, will be effective treatment for OA. -
口腔インプラントの骨結合獲得難易度を予測する生物学的診断法の開発
Grant number：15659463 2003 - 2004
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
窪木 拓男, 高柴 正悟, 滝川 正春, 荒川 光, 藤沢 拓生
Grant amount：\3300000 （ Direct expense: \3300000 ）
1.チタンの細胞培養および遺伝子発現への影響
骨芽細胞様細胞株(MC3T3-E1細胞)の細胞培養培養および遺伝子発現に対するチタンの影響を検討した。
1)チタンプレート
ポリスチレン製の培養皿と表面粗さを同程度にするために,研磨ガラスにチタンを真空蒸着したものを使用した。
2)細胞接着への影響
通常の培養皿と比較してチタンは細胞接着を抑制する傾向にあった。
3)細胞増殖への影響
通常の培養皿と比較して,細胞播種後1,2日ではチタンでは増殖が抑制されるものの3日では両材料ともコンフルエントに達した。
4)細胞分化への影響
骨芽細胞の分化の指標のひとつであるアルカリホスファターゼ活性は,両材料ともに細胞がコンフルエントになった後5日目ごろより上昇し,14日目でピークを向え,21日目では低下した。チタンでは通常の培養皿と比べてアルカリホスファターゼ活性は抑制された。
5)遺伝子発現への影響
通常の培養皿と比較し,チタンの遺伝子発現への影響をサブトラクティブハイブリダイゼーション法にて検討したところ,両材料間で発現に差のあるsod-1,xab-2の遺伝子を検出した。
6)リアルタイムPCR法による遺伝子発現の変動
サブトラクティブハイブリダイゼーション法にて検出した発現に差のあるsod-1,xab-2の経時的な発現の変動を検討したところ,培養皿では細胞播種後5日目で発現のピークを向え,その後低下した。チタンでは発現のピークが10日目前後と培養皿より遅延し,発現も抑制されていた。 -
Development of chair-side assay system for ongoing bone loss with peri-implant sulcus fluids
Grant number：15592049 2003 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
ARAKAWA Hikaru, KUBOKI Takuo, KANYAMA Manabu, KOJIMA Shunji
Grant amount：\3300000 （ Direct expense: \3300000 ）
The first aim of this study was to correlate MMP-1,MMP-8 and MMP-13 in the PISF samples collected from ongoing bone loss sites around per-implants with the periodontal treatments using Western blotting and to determine expression of MMP-8 by immunohistochemistry. Four peri-implantitis patients and four healthy subjects (control group) were selected from a sample of 64 consecutive patients who had attended the Okayama University Hospital from 1990 to 2000. Initial positive test was observed only in three patients. Following the first sampling, two patients received peri-implant mechanical and anti-microbial treatment, while one patient received no active treatment because of his inability to attend the hospital regularly. At the second PISF sampling analysis, an 85 kDa band corresponding to pro-MMP-8 was observed only in one patient received no active periodontal treatment. Half a year following the second PISF sampling, the implant in this patient unfortunately was lost because of severity of peri-implantitis. And MMP-8-like immunoreactivity was clearly seen around inflammatory connective tissue cells in from active peri-implantitis patients.
The next aim of this study was to identify the risk factors in acquisition and maintenance of osseointegration using multivariate analysis. Subjects were consecutive patients who had been installed and finished the secondary surgery osseointegrated dental implants in our hospital from February 1990 to March 2002. Based on the adjusted multivariate models, tobacco use, jaw and implant coating were statistically associated with acquisition of osseointegration. While the risk factors in maintenance of osseointegration were jaw and implant length. -
Gene Therapy for Periodontal Diseases -Regulation of host response by local gene delivery-
Grant number：14370710 2002 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
TAKASHIBA Shogo, KUBOKI Takuo, NISHIMURA Fusanori, KUBOTA Satoshi, MYOKAI Fumio
Grant amount：\13500000 （ Direct expense: \13500000 ）
Because understanding the target factor was important, we decided to specify the target factor from both sides of non-specific host defense and tissue regeneration. Moreover, because it was also anxiety against clinical application of gene therapy because of its toxicity, the experiments are performed for testing it.
1.Target gene
In the rat, cytochrome c oxidase gene expressed strongly at 1 week and pro-α-2 type I collagen gene expressed strongly at 2.5 weeks after alveolar bone begun regeneration. Activation of these genes seem to be needed for alveolar bone regeneration. In dental pulp would, the homolong of human 12.7K-interacting protein 2 expressed strongly. We named it rat FIP-2 gene. It is under analyzing now for physiological meaning. In addition, inflammation, promoter region required for LPS-induced transcription of LITAF was revealed, which is new transcription factor for human tumor necrosis factor(TNF)-α.
2.Introduction of β-defensin by non-viral vector
Anti-bacterium peptide β-defensin gene was transferred to human epithelial cells and rat salivary glands to test its effect for reduce bacteria around cultured cells or rat oral cavity. Furthermore, its effect for local tissue inflammation was also examined. We found that bacterial numbers are reduced and that no obvious inflammation in rat salivary gland tissue. However, when electroporation was used for gene delivery, obvious inflammation was detected. Furthermore, It was revealed that β-defensin gene transcription requires 2 regions of NF-kB binding domain on its promoter, but that NF-IL6 biding domain on it acts to reduce its transcription. -
Establishment of a autologous cell transplantion method using mesenchymal stem cells for perio-dontal tissue and alveolus bone regeneration.
Grant number：14370632 2002 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
KUBOKI Takuo, UEDA Minoru, TAKIGAWA Masaharu, TAKASHIBA Shogo, MAEKAWA Kenji, YOSHIDA Yasuhiro
Grant amount：\14800000 （ Direct expense: \14800000 ）
1.Isolation human bone marrow cells and cell culture.
Bone marrow cells were isolated from human iliac bone marrow of volunteer. Human bone marrow cells were plated and cultured in DMEM containing 10% FBS. Culture medium was changed every 3 days, and their multipotent (osteoblastic and adipogenic) differentation abilities were confirmed
2.Connective tissue growth factor (CTGF/CCN2) enhanced hMSC attachment, migration and survival in a hydroxyapatite scaffold
Human bone marrow cells were incubated to attach onto porous HA blocks for a week. The porous HA/cells hybrids were implanted subcutaneously in nude mice (4 week-old) with CTGF (1ug) or distilled water (control).
The implants were harvested after 4 weeks for SEM observation. SEM observation supported that hBMSC-like cells migrated and survived inside of the porous HA scaffold with CTGF application, while without CTGF, no viable cells were observed inside of the scaffold.
3.hMSC initial attachment and proliferation enhanced on titanium
Adsorption of poly phosphoric acid to Ti disk surface was achieved by immersing Ti disk poly phosphoric acid solution (1 wt%) for 24 hours. Adsorption of polyphosphoric acid onto Ti disks enhanced attachment and proliferation of hMSC.
4.Effect of gene expression of osteoblast on titanium
Osteoblast was cultured on titanium dish and searched a titanium specific gene by cDNA subtractive hybridization. It became clear on titanium that sod-1, gene expression of ribosomal protein L19 were restrained significantly. -
Development of the methods for application of the factors that biologically accelerate reparative dentin formation
Grant number：14571844 2002 - 2003
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
SONOYAMA Wataru, TAKIGAWA Masaharu, TAKASHIBA Shougo, KUBOKI Takuo
Grant amount：\3900000 （ Direct expense: \3900000 ）
1. Pulp cell isolation from human extracted tooth and confirmation of their phenotype
Under permission of ethical committee, pulp cells were isolated from human extracted tooth. RT-PCR was carried our to confirm their gene expression profile and phenotype. As a result, they expressed odont oblast-specific gene, dentin sialophosphoprotein (DSPP), and were suspected to be odont oblast lineage.
2. Effects of Growth factors on their attachment, proliferation, and differentiation
Effects of growth factors, e.g., transforming growth factor-beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), and connective tissue growth factor (CTGF), on their attachment to plastic dish were investigated. As a result, adsorption of these growth factors enhanced their attachment to plastic dish. Effects on their proliferation and alkaline phosphatase (ALPase) activity were also investigated. Concerning about proliferation, only TGF-beta1 enhanced their proliferation. While ALPase activity was downregulated by TGF-beta1 and bFGF.
3. Effects of hydroxyapatite (HAP) on their attachment
To estimate compatibility of HAP with pulp-derived cells, their attachment onto HAP was investigated. As a result, attachment onto HAP was significantly higher compared to plastic dish made of polystyrene. Adsorption of growth factors onto HAP tended to enhance attachment, but the difference was not significant.
4. Effect of HAP on their gene expression
To estimate that HAP have an effects on gene expression of pulp-derived cells or not, they were seeded onto HAP and their gene expression were investigated by RT-PCR. As a result, DSPP and type 1 collagen gene expression were significantly enhanced. -
Molecular mechanism of temporomandibular joint osteoarthritis and gene therapy for degraded, cartilage
Grant number：14571843 2002 - 2003
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
FUJISAWA Takuo, TAKIGAWA Masaharu, NISHIDA Takashi, KUBOKI Takuo, MAEKAWA Kenji
Grant amount：\4000000 （ Direct expense: \4000000 ）
The purposes of this research are to clarify the mechanism of cartilage degradation in osteoarthritis (OA) and to investigate the possibility of gene therapy for articular cartlige restoration using connective tissue growth factor (CTGF).
First, we investigated the effects of CTGF/Hcs24 transduced by recombinant adenoviruses on the rabbit articular cartilage (RAC) cells in vitro. When RAC cells were infected with adenoviruses containing the CTGF/Hcs24 gene, RAC cells expressed CTGF/Hcs24 mRNA and produced CTGF/Hcs24 protein. RAC cells synthesized more proteoglycan than the control cells. These results suggest that CTGF is useful factor for cartilage repair.
Second, we establish a genuine mechanical-stress-induced OA model of the rabbit TMJ. In the experimental rabbits, repetitive forced jaw opening (RFJO) 3 hours/day for 5 days was applied. By histological assessment of the TMJ articular tissues, partial eburnation of the articular cartilage, reactive marginal proliferation of the articular cartilage chondrocytes and nested proliferation of chondrocytes in the subchondral bone area were observed at 7 days after the RFJO period. Furthermore, apoptotic chondrocytes were observed in the cartilage degradation area at 7 days after the RFJO period. And nitrotyrosine, a marker of NO production, and MMP-3, a key factor of cartilage ECM degradation, were observed where chondrocyte apoptosis was evident. These results suggest the RFJO protocol without any surgical intervention can induce evident OA-like lesions in the rabbit TMJ, and cartilage degradation in OA may be induced via chondrocytes apoptosis. This OA model may greatly contribute to the elucidation of the cartilage degradation mechanism in TMJ OA. -
Responses of Hypothalamic-Pituitary-Adrenal axis to cold pressor stimulation
Grant number：13672032 2001 - 2002
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
SUZUKI Koji, KASAI Teruo, MAEKAWA Kenji, KUBOKI Takuo
Grant amount：\3500000 （ Direct expense: \3500000 ）
Objective : Functional abnormalities of Hypothalamic-Pituitary-Adrenal (HPA) axis have been known implicated in chronic pain conditions (e.g., fibromyalgia). This study evaluated the changes in plasma endogenous opioid, HPA hormones concentration and trigeminal heat pain threshold upon cold pressor (CP) stimulation. Methods : In CP trials, ten healthy males (mean age : 25.3^^+__-1.3yrs) immersed their right hands for 2 minutes in 4℃ cold water. Mock trials were also done without the cold water. Whole blood samples were collected 5 minutes before, during, and 5, 15, 30, 45, 60 minutes after CP and mock stimulation for measuring Corticotropin-Releasing-Factor (CRF), Adrenocorticotropic hormone (ACTH), β-endorphine and cortisol. Whole blood was centrifuged for plasma separation, then frozen (-20℃) for later assay. Plasma CRF, ACTH, β-endorphine and cortisol concentrations were determined using radioimmunoassay techniques. Heat pain thresholds in subjects' right V2 region were simultaneously recorded 5 minutes before and 5, 30, 60 minutes after CPS using Thermal Sensory Analyzer (Medoc, Israel). Results : Mean plasma ACTH and β-endorphine levels significantly increased 5 minutes after cessation of CP stimulation and returned to the baseline in 60 minutes (p<0.01, One-way repeated measure ANOVA). Mean plasma cortisol levels also increased 30 minutes after cessation of CP stimulation (p<0.01), while no plasma CRF change was detectable (p=0.83). Mean heat pain thresholds did not show statistically significant changes during the experimental time course (p=0.41), since 4 of 10 subjects showed no obvious change in the heat pain thresholds even with the same CP stimulation schedule. Meanwhile, no variables observed in this study showed a significant change in the mock trial. Conclusions : These results suggest that CP stimulation induces progressive increase in plasma endogenous opioid and HPA hormones levels in healthy males, but this acute plasma endogenous opioid increase does not always associate to heat pain threshold responses in V2 skin region.
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MMP-3転写調節部位遺伝子多型からみた顎関節症の予後に関する分子遺伝学的研究
Grant number：13672033 2001 - 2002
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
水口 一, 滝川 正春, 矢谷 博文, 窪木 拓男, 藤澤 拓生
Grant amount：\4000000 （ Direct expense: \4000000 ）
MMP-3遺伝子のプロモーター領域の多型(5A/6A)と関節疾患感受性との関連性を明らかにすることを目的として遺伝子多型解析を検討した.まず,本年度は遺伝子多型解析に先立ち,本研究計画に対して岡山大学歯学部倫理委員会の承認を得る必要があった.そのため,平成13年11月から岡山大学歯学部倫理委員会の承認を受けた後,岡山大学歯学部附属病院第一補綴科に顎関節部の疼痛,機能障害を主訴に来院し,本研究の趣旨を文書にて説明し自発的同意の得られた被検者から一律3mlの血液採取を行った.
平成14年1月30日現在での被検者数は,男性18名,女性26名の計44名であり,これら被検者の血液よりDNAの抽出を行った.このDNA抽出に関する手法は確立し得た.
現在,抽出されたDNAから、MMP-3プロモーター領域の対立遺伝子の多型(variable number of tandem repeat : VNTR)ならびにIX型コラーゲンα3鎖の遺伝子多型(SNPs)を検討すべく,Ye et al.(1995)の方法ならびにPaassiltaらの方法に従い、PCR産物に対してTthlllI酵素処理を応用し,被検者個々の対立遺伝子の多型を明らかにした.
その結果,現在集積された被検者数では,遺伝子多型と関節疾患感受性との間に統計学的に有為な関連性を見いだすことはできなかった.しかしながら,現段階では被検者数が少なくtype II errorが生じている可能性があり,今後も被検者数の継続的蓄積が必要となると考えられた. -
血管径調節関連遺伝子多型からみた慢性筋痛の分子遺伝学的検討
Grant number：13877327 2001 - 2002
日本学術振興会 科学研究費助成事業 萌芽研究 萌芽研究
窪木 拓男, 矢谷 博文, 鈴木 康司, 前川 賢治
Grant amount：\2000000 （ Direct expense: \2000000 ）
全身の多部位に慢性筋痛を訴える線維性筋痛症(FMS)患者のβ_2アドレナリン受容体(β_2AR)機能評価のため,末梢血由来単核球細胞上に存在するβ_2ARを用い,情報伝達の際に産生されるcAMPを機能性マーカーとして,ELISA法により測定し,年齢,性別をマッチングさせた正常被験者のそれと比較した.FMS群(女性8名,平均年齢44.3歳)と無症状群(女性9名,平均年齢35.8歳)から静脈血を採取し,単核球を分離した.分離した単核球は10^6個ずつ分注し,安静時ベースラインと10^<-3>,10^<-5>Mに希釈したβAR刺激薬(Isoproterenol, IP)を5分間作用させた低・高濃度IP刺激後の3条件のcAMP量を測定した。その結果,低濃度である10^<-5>M IP作用後のcAMP量は,無症状群では有意な増加を示したが,FMS群では変化がみられなかった(無症状群:p<0.001,FMS群:P=0.520).FMS群において単核球におけるβ_2ARの刺激に対する反応が抑制されているという事実は,FMSの病態に全身性のβ_2AR機能の脱感作が関与する可能性を示唆する重要な所見と思われた.一方で,FMSとの病態メカニズムの差異が論争されている慢性局所性筋痛症のβ_2AR機能評価を,慢性筋痛者11名,正常被験者21名を対象にして前述の手法に従って行った.その結果,単核球が産生するcAMP濃度はIP刺激濃度依存性に増大したが,その濃度変化量は両群間に有意な差が見られなかった.本結果は,局所性慢性筋痛症の病態には,全身性のβ_2AR機能異常の関与は認められないこと,換言すれば,線維性筋痛症と局所性慢性筋痛症の病態が異なることを示す重要な所見であると考えられた.遺伝子多型の解析については「岡山大学歯学部ヒトゲノム・遺伝子研究倫理審査委員会」より患者の遺伝子解析について承認を得た後,本学歯学部附属病院顎関節症・口腔顔面痛み外来を受診した患者のなかで,本研究計画の被験者選択基準に合致し,研究の参加に同意の得られた者より,末梢血3mlを供与してもらい,サンプル数を増加させ,解析を進めている.
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Risk assessment of peri-inplantitis patients with chair-side MMP-8 assay system
Grant number：13672031 2001 - 2002
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
ARAKAWA Hikaru, KASAI Teruo, YATANI Hirofumi, KUBOKI Takuo
Grant amount：\4000000 （ Direct expense: \4000000 ）
Matrix metalloproteinases (MMPs) are known to play an important role in pathological tissue destruction in the periodontal tissues. Therefore, MMPs related to bone matrix degradation could be a possible marker for ongoing bone destruction around the peri-implantitis implants. In this study, four peri-implantitis patients (male/female : 2/2 ; mean age : 68.5^^+__-6.7 yrs ; 9 implants ) and age- and sex- matched four control patients (male/female : 2/2, mean age : 66.0^^+__-5.8 yrs, 8 implants) were selected from 64 consecutive patients who were installed implant-supported superstructures in our school from 1990 to 2000. Inclusion criteria for selection of the peri-implantitis and control patients in this study were having an implant "with accumulated vertical bone loss (ABL) of more than 0.6 mm per year" and "with ABL of less than 0.2 mm per year" after superstructure installation, respectively. Bone levels around implants were assessed by dental x-ray films taken annually and mean values of three calibrated observers readings were used as outcome. Ongoing vertical bone loss per year (OBL) after superstructure installation was also calculated. Gingival crevicular fluids (GCFs) were collected at the mesio-buccal comer of the gingival crevices by using absorbent paper points. Then the samples were dissolved in 0.1% phoshate buffered saline with Tween 20 (PBS-T) and stored at -20℃. MMPs in GCFs were detected by Western blotting using anti-human monoclonal antibodies specific for MMP-1, -8 and -13 (Fuji Chemical Industry, Japan) with positive controls. As the results, in the peri-implantitis patients, MMP-8 was detected in three actively bone-resorbing implants whose OBL at GCF sampling was more than 0.8 mm and not detected in the other six implants whose OBL at GCF sampling was less than 0.3 mm, while no MMP-8 was detected in the controls. MMP-1 and -13 could not be detected in any GCFs in this study. These results suggest that MMP-8 might be a marker for prediction of ongoing bone destruction around the peri-implantitis implants.
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Gene therapy for temporomandibular joint osteoarthritis
Grant number：12557169 2000 - 2002
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
KUBOKI Takuo, NAKANISHI Tohru, TAKIGAWA Masaharu, FUJISAWA Takuo, KASAI Teruo, YATANI Hirofumi
Grant amount：\12000000 （ Direct expense: \12000000 ）
The purposes of this research are to clarify the mechanism of cartilage degradation in osteoarthritis (OA) and to investigate the possibility of gene therapy for articular cartlige restoration using connective tissue growth fector (CTGF).
First, we establish a genuine mechanical-stress-induced OA model of the rabbit TMJ. In the experimental rabbits, repetitive forced jaw opening (RFJO) 3 hours/day for 5 days was applied. By histological assessment of the TMJ articular tissues, partial eburnation of the articular cartilage, reactive marginal proliferation of the articular cartilage chondrocytes and nested proliferation of chondrocytes in the subchondral bone area were observed at 7 days after the RFJO period. These results suggest the RFJO protocol without any surgical intervention can induce evident OA-like lesions in the rabbit TMJ, and this OA model may greatly contribute to the elucidation of the cartilage degradation mechanism in TMJ OA.
Second, we investigated the effects of CTGF/Hcs24 transduced by recombinant adenoviruses on the rabbit articular cartilage (RAC) cells in vitro. When RAC cells were infected with adenoviruses caontaining the CTGF/Hcs24 gene, RAC cells expressed CTGF/Hcs24 mRNA and produced CTGF/Hcs24 protein. RAC cells synthesized more proteoglycan than the control cells. -
Molecular cloning of the factors that biologically accelerate reparative dentin formation
Grant number：12470418 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
KUBOKI Takuo, TAKIGAWA Masaharu, TAKASHIBA Shougo, SONOYAMA Wataru, KANYAMA Manabu, NAKANISHI Tohru
Grant amount：\13800000 （ Direct expense: \13800000 ）
1. Gene delivery to cultured cells
We prepared recombinant adenovirus vector carrying beta-galactosidase gene. Mouse osteoblast-like cells, MC-3T3 -E1, were cultured with this vector. As a result, almost all cells were transfected with beta-galactosidase gene.
2. Localization of known factors (TGF-beta 1 and CTGF) in vivo animal model
Localization of TGF-beta 1, that is supposed to be involved in reparative dentinogenesis, and CTGF were confirmed with immunohistochemical staining in animal (wister rat) experimental model. As a result, in 2 weeks from tooth reduction reparative dentin-like tissues were observed, and strong staining of TGF-beta 1 and CTGF were observed around these tissues.
3. Gene expression of TGF-beta 1 and CTGF in cultured cells stimulated with proinflammatory factors
MDPC-23, mouse-derived odontoblast-like cells were used in this study. The cells were stimulated with IL-1 beta and bacterial LPS. Changes in CTGF and TGF-b1 genes expression were examined by RT-PCR. As a result, MDPC-23 cells were expressing the CTGF and TGF-beta 1 genes coastitutively, and both factors increased CTGF gene expression and decreased TGF-b1 gene expression in the odontoblast-like cells (MDPC-23) within one-day period after stimulation.
4. Effect of TGF-beta 1 and CTGF to cultured cells
The effects of rCTGF and rTGF-beta 1 on cell proliferation were determined by the MTT assay. rTGF-beta 1 tended to decrease the proliferation dose-dependently, whereas effect of rCTGF was not evident. Next, to investigate the effects of rCTGF on calcification of MDPC-23 cells, cells were cultured with medium containing rCTGF. Sequential addition of AA and b-GP up-regulated the ALPase activity, while addition of rCTGF had no obvious effects. -
MRI brain mapping image analysis of oral and maxillofacial region by various sensory stimuli
Grant number：12671825 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
KISHI Kanji, SHIGEHARU Hiroshi, WAKASA Toru, SUGIMOTO Tomosada, KUBOKI Takuo
Grant amount：\3400000 （ Direct expense: \3400000 ）
The aim of this study was to decide the adequate pulse sequence suitable for fMRI study of various sensory stimuli, and to establish the fixation of the head and image processing method, first of all. Secondly, we applied fMRI to the healthy volunteers on the tasks of gustatory stimulation and swallowing, and investigated the possibility of fMRI in the oral and maxillofacial region on clinical application.
In 2000, first of all, we investigated the scanning method. We performed fMRI study of hand-grasping task by two pulse sequences, gradient echo type EPI(GE-EPI), and spin echo type EPI(SE-EPI), and compared the fMRI images by these two sequences. As a result, the specificity and accuracy of the signal detectability by SE-EPI were higher than those by GE-EPI. Therefore, we decided the SE-EPI sequence on the following study.
The fixation of the head was performed by applying the hair-band and sponge to the head-coil and the head of the subjects. Visual stimulation and auditory stimulation were shut by using eye-mask and ear-plug.
Image processing was performed by the software (Numaris) included in Magnetom Vision, and fMRI images were produced by the statistical calculation using z-score.
Then, fMRI study by gustatory stimulation was performed. We used two tastants ; 1M NaCl and 3mM saccharin. These tastants were dropped into the oral cavity of the subjects through the tube, and fMRI scanning was done. As a result, pariental operculum, frontal operculum, and insula were mainly activated. There were no differences of activated areas between NaCl and saccharin.
In 2001, fMRI by swallowing task was performed. Distilled water was injected into the oral cavity of the subjects at 3ml/sec. The subjects swallowed the water intermittently. As a result, primary motor cortex, and primary somatosensory cortex were mainly activated. -
Molecular biological study on pithophysiology of chronic muscle pain from the view of abnormal beta2-adrenergic receptor function.
Grant number：12671883 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
MAEKAWA Kenji, MINAKUCHI Hajime, SUZUKI Kouji, KUBOKI Takuo, YATANI Hirofumi
Grant amount：\3400000 （ Direct expense: \3400000 ）
The purpose of this study was to investigate alteration of the beta-adrenergic receptor (BAR) in fibromyalgia and chronic localized myalgia patients. Since the BARs are present on circulating lymphocytes and activation of the G-protein coupled receptors like BAR leads to an increase in the intracellular level of cyclic adenosine monophosphate (cAMP), we measured cAMP levels to assess indirectly the functional status of the receptor. Thirty cc peripheral blood samples were drawn from subjects' anterocubital vein. Lymphocyte cells were isolated from the total blood by using the Ficoll-Hypaque gradient technique. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay using a commercially available kit (cAMP EIA system, Amersham, England). Aliquots of even amount of cells were incubated with or without stimulation of beta-agonist isoproterenol (ISO) for 5min. Results : Basal intracellular cAMP levels were not significantly different between FM patients and healthy controls and between localized myalgia and healthy controls. However, beta-agonist induced mean intracellular cAMP increase was significantly reduced in FM patients compared with controls. Regarding the localized myalgia patients, beta-agonist induced mean intracellular cAMP increase was almost identical with that of controls (p=0.806). These results suggest that diminished βAR function might be related to FM pathophysiology. In addition, these findings indirectly support the notion that the pathophysiology of fibromyalgia and localized myalgia are different.
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Effect of mastication on 3-dimensional brain hemodynamics measured by functional MRI
Grant number：12557170 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
MAEKAWA Kenji, KISHI Kanji, SUZUKI Kouji, KUBOKI Takuo, WAKASA Touru
Grant amount：\6500000 （ Direct expense: \6500000 ）
The original purpose of this study was not achieved, because mastication provoked motion artifact to image brain hemodynamics using MRI. For this reason, we were forced to change the research protocol and investigated the cerebral cortical activation during volitional swallowing using functional MRI. The results showed that the swallowing task activated the inferior parts of the pre- and postcentral gyri. Additionally, we also investigated the correlation between T2 weighted MRI signal intensity (S1) and intramuscular blood volume in human muscle. First, we compared blood volume changes transcutaneously measured using near-infrared (NIR) spectroscopy against SI changes taken from a transverse T_2-weighted MR image of the masseter muscle in healthy human subjects before, during and after contraction. The data showed that both NIR-based total blood volume and T_2-weighted MRI-based SI levels clearly decreased during muscle contraction and a clear post-contraction rebound response was evident after the contraction. The NIR data were found to be highly correlated to MRI-based SI data (Pearson's r=0.945, p<0.0001). Next, we evaluated the ability of SI in T2-weighted trapezius muscle MRI to detect intramuscular hemodynamic changes upon cold pressor stimulation (sympathetic nerve activator). Cold pressor stimulation (4℃) was applied to each subject's right foot and ankle for 2 min. The SI changes were recorded continuously for 7 min before, 2 min during, and 6 min after withdrawal of cold pressor stimulation. The mean SI level in T2-weighted trapezius muscle MRI significantly increased during cold pressor stimulation (p<0001, one way repeated measure ANOVA and post hoc contrast analysis) and returned to the baseline level after cold pressor withdrawal. These findings are identical to the cold pressor-induced hemodynamic changes documented in the trapezius muscle by NIR spectroscopy evaluation (Acero et al., 1999). Both studies provided the evidence that SI measurement in T2-weighted muscle MRI is sufficiently sensitive to detect intramuscular hemodynamics in humans.