口腔扁平上皮癌の浸潤増殖動態に関する研究

金沢大学医学部研究代表者の山本は1983年に新しい浸潤様式分類を発表して以来、口腔扁平上皮癌の浸潤機序の解明を目的とした実験的研究を系統的に行い、in vitroモデルやin vivoモデルについての成果の一端を既に発表してきた。本報告書ではin vivoモデルでの浸潤像について免疫組織学的ならびに基質分解酵素の活性をゼラチンザイモグラフィにて検討したので報告する。移植を行った癌細胞は5種類のヒト口腔扁平上皮癌細胞で、OSC-19、OSC-20、SS、FA、TT細胞を使用した。移植方法は癌細胞を培養液中で8×10^6個/mlの濃度に調整し、ヌードマウスの口腔内に移植した。口腔内移植腫瘍の組織像は各々の細胞の由来する原発腫瘍でのものと類似していた。頸部リンパ節転移はOSC-19細胞が81%、TT細胞が50%に認められ、この2つの細胞は浸潤様式が4C型を呈していた。IV型コラーゲンの免疫染色の結果では、リンパ節転移がみられなかったFA細胞では胞巣周囲に連続線状に認められたが、その他の転移のみられた細胞では基底膜の断裂や消失が観察された。デスモグレイン(DG)の免疫染色の結果では、FA細胞では腫瘍細胞間にDG陽性部分を認めたが、その他の細胞ではDGの減弱が観察された。浸潤部のPCNA陽性率の検討を行ったが、浸潤像との明らかな相関は認められなかった。マトリックスメタロプロテナーゼ(MMP)の染色結果では、最も高浸潤性のOSC-19細胞のみがMMP-1とMMP-9の双方とも陽性であった。ゼラチンザイモグラムで培養上清に含まれる酵素を調べると、OSC-19細胞ならびにOSC-20細胞のすべてに72kDaと92kDaの位置にゼラチン分解活性を示すバンドが見られたが、このバンドの強度はOSC-20細胞に比較してOSC-19細胞が明らかに強く観察された。以上の結果より、口腔扁平上皮癌における個々の浸潤像は癌細胞と間質あるいは癌細胞同士の相互関係が重要な因子として影響を及ぼしている可能性が示唆された。We devised a new model in order to establish an in vivo model for human oral carcinoma that exhibits significant local invasion and metastasis. In the present study, we examined the invasive sctivity of five kinds of oral squamous carcinoma cells (OSC-19, OSC-20, SS, FA and TT) by this new model. The nude mice had tumor cells from one of five oral squamous cell carcinoma cells implanted into the tongue or the oral floor via an intra-oral route and, as a control, the subcutaneous tissue of the back. The back tumors showed a benign growth pattern, lacking significant invasion of surrounding tissues. In contrast, the tumors implanted into the tongue or the oral floor exhibited invasive growth and stromal reactions, the histological appearance of which was similar to that of the original tumors. Moreover, the regional neck lymph node and pulmonary metastases were observed in this model. Regional neck lymph node metastases were detected in 81.0% of mice implanted with OSC-19 cells. The oral tumor implanted with OSC-19 cells or TT cells showed a cords-like diffuse invasion pattern and was characterized by the abscence of a continuous basement membrane and by a reduction in the intercellular adhesion of carcinoma cells. These results suggest that the invasion of oral squamous cell carcinoma may be influenced by the tumor-stromal and/or intercellular interactions.研究課題/領域番号:06454567, 研究期間(年度):1994 – 1995出典：研究課題「口腔扁平上皮癌の浸潤増殖動態に関する研究」課題番号06454567（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-06454567/064545671995kenkyu_seika_hokoku_gaiyo/）を加工して作

口腔扁平上皮癌の浸潤増殖動態に関する実験的研究

金沢大学医学系研究科本研究課題ではin vitroおよびin vivoでの各浸潤モデルにおける浸潤増殖像の検討およびin vivoでは浸潤モデルにおける浸潤像と転移との関係についてそれぞれ検討した。その結果、コラーゲンゲルを用いたin vitro浸潤モデルにおいて各癌細胞の浸潤能に応じた。浸潤像を再現することに成功し、浸潤様式が高度になるにつれゲル中へのび漫性の浸潤を認めた。また、浸潤様式4D型の癌細胞ではコラーゲンゲル中に繊維芽細胞を入れない状態でも、同様にび漫性の浸潤を認めた。.これらの細胞の運動能を検討した結果、浸潤能の高い細胞で浸潤時に自己分泌型の運動促進因子(autocrine motility factor ; AMF)の高い発現を観察し、高浸潤細胞では繊維芽細胞に頼らず自ら産生する運動因子で浸潤する能力があることが証明された。基質分解能では各種のマトリックスメタロプロテナーゼ(MMP)を検討したところ浸潤能の高い細胞でMMP-2,MMP-9,MT1-MMPの過剰発現を認めた。接着能では細胞間接着分子であるカドヘリンおよびデスモグレインについて検索したところ、浸潤能の低い細胞の浸潤像では細胞間接着が強固に観察されたが、浸潤能の高い細胞の浸潤像では両者の接着分子はいずれも高度に消失していた。以上より口腔扁平上皮癌の浸潤像の形成には運動能、基質分解能、接着能が密接に関係していることが示唆された。そこで、同じ癌細胞をヌードマウスの口腔内に移植した正所性移植モデルにおける転移能を検討したところ、高度浸潤像を示す細胞では100%のヌードマウスに頸部リンパ節転移を認めた。また、in vivoにおいても、浸潤像の形成には運動能、基質分解能、接着能が密接に関係していることが示唆された。さらに、リンパ節転移においても同様に、これらが密接に関係している結果を得ている。Since 1978, we have been studying the mode of cancer invasion as the important prognostic factor in patients with oral squamous cell cancer. Grade 4 of mode of invasion proposed by Jakobsson was sub classified into grades 4C (Cord-like type) and 4D (Diffuse type). The purpose of this study is to immunohistochemically and experimentally elucidate this modified grading system for the mode of invasion. Materials and methods used are human cancer materials, DMBA induced tongue cancer and in vitro and in vivo invasion model by using 3 cell lines; OCS-20 (Gr.3), OSC-19 (Gr. 4C) and HOC-313 (Gr. 4D). In immunostained human cancer materials, the higher the grade of the mode of invasion was, the less continuous the basement membrane was, and the less intercellular adhesion was. Majority of diffuse invasive cancers (Gr. 4C and 4D) co-expressed membrane type- matrix metalloproteinase and matrix metalloprotenase-2. In cell motility assay, HOC-313 showed the greatest motility (autocrine motility factor; AMF). Cell lines cultured on 3T3 fibroblast embedded collagen gels showed similar invasion pattern to their origin. Among them, only HOC-313 showed invasion even if fibroblast was not embedded. In DMBA induced tongue cancer, the mode of invasion were observed from Gr.1 to Gr.4C. OSC-20 and OSC-19 implanted orthotopically in the tongue of nude mice grew and metastasized to lymph nodes. Their histologic appearance was similar to that of the origin. HOC-313 grew temporarily for several months by support of matrigel. In recent study, cell selected TSU (Gr.4D) produced lethal tumorgenicity in SCID mouse with similar histology to the origin. This grading system was also proven immunohistochemically and experimentally justifiable.研究課題/領域番号:11470432, 研究期間(年度):1999 – 2001出典：「口腔扁平上皮癌の浸潤増殖動態に関する実験的研究」研究成果報告書　課題番号11470432（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-11470432/）を加工して作

口腔扁平上皮癌の浸潤・転移における腫瘍間質および血管新生の意義

金沢大学医薬保健研究域医学系癌細胞が浸潤する際にはその周囲の線維性結合織や血管の増生を伴うことが知られている。また、口腔扁平上皮癌においては浸潤様式4C型の癌では胞巣周囲に多量の線維性結合織が観察される。しかし、この間質の反応は腫瘍の浸潤にとってどのような意義があるのか不明である。そこで、線維芽細胞増殖抑制剤による口腔扁平上皮癌の浸潤や転移に対する効果を検討した。実験には浸潤様式4C型の口腔扁平上皮癌細胞株OSC-19と線維芽細胞株Swiss-3T3を用いた。線維芽細胞増殖抑制剤はすでに臨床に用いられているトラニラストを使用した。はじめに、各細胞のトラニラストに対する感受性を検討するため、MTT assayを行った。また、癌細胞の浸潤と転移に対する効果を検討するため、ヌードマウスの舌にOSC-19細胞を移植し、トラニラストの腹腔内投与を行い、効果を検討した。MTT assayの結果、3T3細胞はトラニラストの濃度依存的に増殖抑制効果を認めたが、OSC-19細胞はトラニラスト高濃度下においても増殖抑制効果は認められなかった。また、移植腫瘍においては、トラニラスト投与群は非投与群と比較して腫瘍の大きさは小さく、増殖抑制効果が認められた。したがって、本剤投与によって腫瘍全体の線維芽細胞の量は減少した。病理組織学的には癌細胞の壊死などは認められず、浸潤像にも大きな違いは見られなかった。リンパ節転移率は非投与群100%に対して、投与群は57%であり転移抑制効果を認めた。トラニラスト投与の効果の一つに血管新生阻害があげられる。本剤投与による血管構築の違いについても検討され、血管新生阻害作用が認められた。研究課題/領域番号:14657519, 研究期間(年度):2002 – 2003出典：「口腔扁平上皮癌の浸潤・転移における腫瘍間質および血管新生の意義」研究成果報告書　課題番号14657519（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-14657519/)を加工して作

Use of self-setting α-tricalcium phosphate for maxillary sinus augmentation in rabbit

金沢大学医薬保健研究域医学系Purpose: The purpose of this study was to histologically and immuno-histochemically evaluate tissue changes in the maxillary sinus after bone screw implantation and maxillary sinus augmentation using self-setting α-tricalcium phosphate (α-TCP; BIOPEX®-R) in rabbit. Study design: Adult male Japanese white rabbits (n=15, 12-16 weeks, 2.5-3kg) were used. The sinus lift was made from the nasal bone of a rabbit. Bone screws (Dual top auto-screw®) were implanted into the nasal bone, and after BIOPEX®-R was implanted into the left elevated space (operated side) an atelocollagen sponge (ACS: Teruplug®) was implanted into the right elevated space (control side). The rabbits were sacrificed at 4, 12 and 24 weeks postoperatively, and formalin-fixed specimens were embedded in acrylic resin. The specimens were stained with hematoxylin and eosin. For immune-histochemical analysis, the specimens were treated with bone morphogenetic protein-2 (BMP-2) antibodies. Finally, these were evaluated microscopically. Results: Tight bonding without fibrous tissue continued between the bone screw and BIOPEX®-R, and the rigidity of the bone screw in the nasal bone was retained for 24 weeks in all cases. The area ofnew bone formation increased gradually on both sides; however, there was no significant difference between both sides at 4, 12 and 24 weeks. The number of BMP-2-stained cells on the experimental side was significantly larger than that on the control side after 4 weeks (P=0.0361). Conclusion: This study suggested the usefulness of self-setting α-TCP (BIOPEX®-R) to maintain the rigidity of implanted bone screws from an early period, and the result of BMP-2 expression suggested that BIOPEX®-R could have bone-conductive activity in the maxillary sinus augmentation. © 2010 John Wiley & Sons A/S

Bite Force and Maxillofacial Morphology in Patients With Duchenne-Type Muscular Dystrophy

金沢大学医学部附属病院歯科口腔外科Purpose: The purpose of this study is to evaluate maxillofacial morphology and bite force in patients with severe Duchenne-type myodystrophy. Patients and Methods: The subjects included 24 men (average age, 21.5 years; range, 17 to 30 years) with Duchenne-type muscular dystrophy receiving treatment in National Ioh Hospital. Lateral and axial cephalograms were used to assess the morphology in this study. The maximum bite force on the first molar and the maximum mouth opening distance were measured. Results: The anterior open bite was visualized in most patients on the lateral cephalogram. The upper and lower arch lengths in the patients were significantly smaller than those in the controls (P < .05). In contrast, the upper and lower arch widths in the patients were significantly larger than those in the controls (P < .05). The maximum bite force and maximum mouth opening distance in the patients were significantly lower than those in the controls (P < .05). Conclusion: These results appear to be very useful for improving the care and treatment of patients with Duchenne-type muscular dystrophy. © 2007 American Association of Oral and Maxillofacial Surgeons

口腔扁平上皮癌の浸潤増殖動態に関する研究

金沢大学医学部1) 臨床材科における浸潤像の免疫組織化学的検討IV型コラーゲン,ラミニン,ヘパラン硫酸プロテオグリカンより構成される基底膜成分の連続性と浸潤様式との関連を免疫組織化学的に検討したところ,浸潤傾向の弱い症例では連続性のものが多く見られたのに対し,浸潤傾向が強くなるにつれ不連続や広範囲消失のものが増加し,特に4D型の症例ではいずれも広範囲消失例であった。またデスモソームの構成成分であるデスモグレインIを用いて細胞間接着性を検討したところ浸潤傾向が強くなるほど,細胞間接着性が低下することが明らかになり,特に4D型の陽性細胞率ではわずか0.5%であった。さらにウロキナーゼ型ブラスミノーゲンアクチペーター(uPA),マトリックスメタロプロテテーゼ(MMP)と浸潤組織像との関連を観察したところ,浸潤傾向が強いほど,uPAやMMPが多く発現することが観察された。このように,4C型と4D型で明らかな相違があるものが多く認められた。2) 浸潤と転移の機序に関する実験的研究in vitroでの細胞運動能実験において3型のOSC20,4C型のOSC19および4D型のHOC313の3種類の口腔癌細胞株を用いて,その自己運動能を金コロイド法を用いて検討したところ,4D型細胞株の運動能が他の細胞株に比べ有意に高かった.またゲルを用いたin vitro浸澗モデルでの研究ではOSC20とOSC19細胞では線維芽細胞の存在下でのみ原発巣に類似の癌浸潤像が形成され癌細胞と線維芽細胞の相互作用の必要性が示されたのに対し,HOC313では線維芽細胞が存在しなくても浸潤する強い浸種能が示され,臨床での4D側の強い浸潤性との相関性が示唆された。一方,ハムスター誘発舌癌における浸潤増殖像の検討では浸潤様式1から4C型まで観察されたが,4D型は認められなかった。さらにヌードマウス移植in vivo浸潤転移モデルによる検討では舌や口底にOSC19,OSC20細胞を生所性移植したところ,臨床に極めて類似の浸潤像が確実に形成されることを見いだし,本法が口腔癌の浸潤・転移モデルとして極めて有用であることが示唆された。現在,通常の方法では生着しない4D型細胞株の可移植化に向けて検討を行っている。3) 浸潤増殖像と腫瘍血管構築像に関する検討臨床材料の免疫組織所見に加え,ハムスター実験系およびヌードマウス実験系について腫瘍血管構築像を生体墨汁を用いて検索したところ,浸潤傾向が強いほど,腫瘍深部の血管密度は低く,細い傾向にあるということが示された。この結果より,腫瘍血管は人癌や実験誘発癌の発生・増殖および移植癌の生着・増殖には必須であるが,浸潤や転移には直接的には必要としないものと推察された。The vascular architecture of DMBA (9, 10-dimethyl I, 2-benzanthracene) induced tongue carcinoma in the hamster was examined. Changes in tumor vessels and their significance during growth of invasive oral squamous cell carcinoma was observed. Tongue cancer was induced by abrading the right margin of the tongue of each hamster with an endodontic barbed broach and subsequently applying 1.0 % DMBA dissolved in aceton, three times a week, at the same site. After macroscopic detection of cancer and the signs of cachexia (weight loss, etc.), Indian-ink was infused into the ascending aorta of each animal under general anesthesia. Tissue was harvested from each animal and made into a transparent specimen, 100 p m in thickness, for examination of vasucular architecture. The mode of cancer invasion was Grade I in 8 animals, Grade 2 in 8, Grade 3 in 23, and Crade 4C in 5. Macroscopic tumor growth mode was exophytic in 36 animals and endophytic in 8 animals. Each mode of cancer invasion was associated with characteristic vascular architecture. That is, low invasive cancers (Grade I and 2) showed dendriform vascularization and a marked increase in vessel density as they underwent exophitic growth. Grade 3 invasive cancei shbwed rings of blood vessels forming during the coume of invasion by alveolar tumor foci. Diffusely invasive cancer (Grade 4C) showed destruction of exisdng blood vessels and a decrease in vessel density. As fumor invasion progressed ; the vessel density decreased (p<0.05). The vessel density was significantly lower in cases of exophytic cancer than in cases of endophytic cancer (p<0.01). The PCNA (proliferating cell nuclear antigen) of cancer cells, which is an indicator of tumor proliferation potential, was more frequently positive as tumor invasion bccame more severe. That is, the PCNA positive rate in Grade I or 2 animals differed significantly from that in Grade 3 or 4C animals (p<0.05). The PCNA positive rate decreased as vessel density increased (p<0.05). Thus, the vessel density was not proportional to the tumor proliferation potential. The expression of vascular endothelial growth factor (VEGF) was seen more frequently as tumor invasion progressed (p<0.05), but the expression of VEGF did not correlate with the vessel density. These results suggested that the changes of the tumor vessel were dependent on the mode of cancer invasion and proliferation.研究課題/領域番号:08457544, 研究期間(年度):1996 – 1998出典：研究課題「口腔扁平上皮癌の浸潤増殖動態に関する研究」課題番号08457544（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-08457544/084575441998kenkyu_seika_hokoku_gaiyo/）を加工して作

Simplified stress analysis on the temporomandibular joint in Class III patients with and without mandibular asymmetry using a rigid body spring model

金沢大学医薬保健研究域医学系To cite this article: Ueki K, Takeuchi N, Nakagawa K, Yamamoto E: Simplified stress analysis on the temporomandibular joint in Class III patients with and without mandibular asymmetry using a rigid body spring model Orthod Craniofac Res 2009;12:312-318 Structured AbstractAuthors - Ueki K, Takeuchi N, Nakagawa K, Yamamoto E Objective - Aim of this study was to investigate the differences in stress on the temporomandibular joint (TMJ) between Class III patients with and without mandibular asymmetry using a rigid body spring model (RBSM). Design - Menton (Me), the centre point of occlusal force on the line that connected the bilateral buccal cusps of the second molars and the most lateral, superior and medial points of the condyle were plotted on frontal cephalograms, and stress on the condyles was calculated with the 2-dimensional RBSM program of fortran. Setting and Sample Population - Eighty Japanese patients with diagnosed mandibular prognathism were divided into two groups, a symmetry group and asymmetry group on the basis of the Mx-Md midline position. Outcome measure - The degree (force partition) of the resultant force, the direction (angulation) and displacement (X, Y) of each condyle were calculated. The horizontal displacement vector (u), the vertical displacement vector (v) and rotation angle (θ) of the mandibular body at Menton were also calculated. Results - There were significant differences between the deviated and non-deviated sides of both groups regarding resultant force (symmetry group: p = 0.0372, asymmetry group: p = 0.0054), X (symmetry group: p < 0.0001, asymmetry group: p = 0.0001) and Y (symmetry group: p = 0.0354, asymmetry group: p = 0.0043). For angulation, there was a significant difference between the deviated and non-deviated sides in the asymmetry group (p = 0.0095). Conclusion - The results of this study suggest that difference in stress angulation on the condyles could be associated with asymmetry in mandibular prognathism. © 2009 John Wiley & Sons A/S

The change of stress distribution on the condyle after mandibular setback surgery

金沢大学医学部附属病院歯科口腔外科The rigid-body spring model (RBSM) theory was incorporated into a model as a discrete method for analysing problems of limit, such as the stress distribution on the condyle. The purpose of this study was to evaluate the two-dimensional RBSM for determining stress on the temporomandibular joint (TMJ) in patients after orthognathic surgery. Thirty-two patients (five males and 27 females, mean age 21.4 ± 4.9 years) with mandibular prognathism underwent bilateral sagittal split ramus osteotomy (SSRO) and setback; 48 subjects were recruited as controls. Anatomical landmarks were traced from pre- and post-operative lateral cephalograms and the information was processed using the Fortran analysis program. The force vector on the condyle, its degree, its direction, and the displacement co-ordinates (x, y) and rotation (θ) at the gonial angle were calculated. When muscular power was assumed to be 1, the post-operative degree of the force vector was higher than the pre-operative value (P < 0.05). The X co-ordinate, x, and rotation, q, of the displacement vector in the pre-operative patients with mandibular prognathism were significantly higher than those in the control subjects (P < 0.05). There were still significant differences between the displacement values post-operatively between the patients and controls (P < 0.05). The results suggest that the degree and direction of the force vector and the resulting displacement co-ordinates can be used as parameters in a surgical model. The RBSM may also be useful in evaluating the pre- and post-operative skeletal morphology of jaw deformities. © The Author 2006. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved

Use of the Sonopet ultrasonic curettage device in intraoral vertical ramus osteotomy

金沢大学医学部附属病院歯科口腔外科This study was designed to evaluate the usefulness of the Sonopet UST-2001 (Miwatec Co., Ltd., Kawasaki, Kanagawa, Japan) ultrasonic curettage device, and to assess the outcome after intraoral vertical ramus osteotomy (IVRO). Thirteen Japanese adults (age range 20-41 years, mean age 29.6 years) presented with jaw deformities diagnosed as mandibular prognathism and asymmetry; they all underwent IVRO of the mandible. This procedure was followed by ultrasonic bone curettage using the Sonopet to make a guiding notch or groove in the lateral cortex of the ramus without damaging the vessels and nerves. After surgery, the osteotomy line was evaluated by three-dimensional computed tomography. In all patients, osteotomy with the Sonopet device was achieved safely, with minimal bleeding and no major complications. The distal segment could be moved into its ideal position and all patients achieved their ideal profiles. Ultrasonic bone curettage is a safe method for making a guiding groove, without damage to surrounding tissue, prior to complete IVRO. © 2007 International Association of Oral and Maxillofacial Surgeons

Stress change on the temporomandibular joint in mandibular prognathism subjects with asymmetry after orthognathic surgery

金沢大学医薬保健研究域医学系The purpose of this study was to evaluate changes in stress on the temporomandibular joint (TMJ) in 80 Japanese subjects (21 males and 59 females, mean age 23.7 years) with mandibular prognathism, with and without asymmetry after orthognathic surgery using the rigid bodies spring model (RBSM). The asymmetric group consisted of 40 subjects whose Mx-Md midline was more than 3 degrees. The remaining 40 subjects formed the symmetric group.The geometry of the stress analysis model was based on frontal cephalograms of the subjects. Menton (Me), the centre point of occlusal force on a line connecting the bilateral buccal cusps of the second molars, and the most lateral, superior, and medial points on the condyle were plotted on a computer display and stress on the condyle was calculated with the two-dimensional RBSM program, Fortran. The degree (force partition) of the resultant force, the direction (angulation), and the displacement (X, Y) of each condyle were calculated and the horizontal displacement (u), the vertical displacement (v), and rotation displacement (θ) of the mandibular body at Me were calculated pre- and post-operatively. The data was analysed using paired and unpaired t-tests.For the vertical (v) and rotational (θ) displacement, the post-operative value was smaller than the pre-operative value (v: P < 0.001, θ: P = 0.0063) in the asymmetric group. For angulation and the X-component, the post-operative value was smaller than that pre-operatively on the deviated (angulation: P = 0.0074, X-component: P = 0.0003) and non-deviated (angulation: P = 0.0024, X-component: P = 0.001) side in the asymmetric group. However, there was no significant difference between the pre- and post-operative value for any parameter in the symmetric group.These findings suggest that surgical correction of mandibular prognathism, with and without asymmetry, could induce an improvement in stress balance on the TMJ in the frontal aspect. © The Author 2010