Masaharu Shirakawa

Transforming Growth Factor-beta-1 Reduces Alkaline Phosphatase mRNA and Activity and Stimulates Cell Proliferation in Cultures of Human Pulp Cells

Transforming growth factor-beta-1 (TGF-beta-1) is a potent modulator of proliferation and differentiation in various tissues, and may be involved in the control of dental development and repair. This study was carried out to investigate the effects of TGF-beta-1 on alkaline phosphatase (ALPase) activity and mRNA level, and on DNA content in cultures of human pulp cells. Four lines of pulp cells (P1-P4), isolated from the upper wisdom teeth of four patients, were maintained separately in monolayer cultures in the presence of 10% fetal bovine serum. TGF-beta-1, at 0.1 ng/mL, increased ALPase activity and DNA content in P1 cultures, but not in P2-P4 cultures. In all cultures, TGF-beta-1, at 5 ng/mL, decreased ALPase activity to a very low level, and increased DNA content. Northern analysis showed that human pulp cells synthesized a single species of 2.6-kb liver/bone/kidney-type ALPase, and that TGF-beta-1, at 5 ng/mL, decreased the level of the ALPase mRNA. These results suggest that TGF-beta-1 is a mitogen for human pulp cells, and that it regulates the activity of the universal-type ALPase at the pre-translational level.

Effects of phenolic dental medicaments on prostaglandin synthesis by microsomes of bovine tooth pulp and rabbit kidney medulla

Prostaglandin (PG) synthesis from [1-14C]-arachidonic acid by bovine dental pulp microsomes was stimulated by 0.1-0.5 mM concentrations of p-chlorophenol (PCP) and inhibited by more than 3 mM. Dual effects (stimulation and inhibition) of phenol, PCP, o-, m-, p- and tri-cresol on PG synthesis by rabbit kidney medullary microsomes were observed. Of various compounds tested, eugenol, thymol and guaiacol were the most potent inhibitors; the inhibitory action was reversible. Phenolic compounds did not affect the activity of glucose-6-phosphatase, adenosine triphosphatase, or lactate dehydrogenase in rabbit kidney medulla within the range of concentrations that stimulated or inhibited PG synthesis. Thus the analgesic effect of phenolic medicaments in endodontic therapy may be due to inhibition of arachidonic-acid metabolism.

Susceptibility of diabetic rat aorta to self-deactivation during prostacyclin synthesis

The ability of aortic rings to produce PGI2 markedly decreased in streptozotocin-induced diabetic rats when compared with age-matched controls. Arachidonic acid dose-dependently stimulated the production of PGI2 both in normal and diabetic rat aorta during 5 min incubation. The deficiency in PGI2 production in diabetic rat aorta was temporarily corrected by the addition of 5-20 microM of arachidonic acid. However, when aortic rings were incubated over a period of 10 min in the presence of arachidonic acid, the ability of PGI2 production in diabetic rats markedly decreased. Repeated exposures of aortic rings to arachidonic acid also markedly reduced PGI2 production in diabetic rats. PGI2 production in diabetic rat aorta was inactivated more readily than in normal rat aorta by pre-incubation with t-butyl hydroperoxide. Phenol had a protective effect on incubation-induced inactivation of PGI2 generating activity in diabetic rat aorta. Serum markedly stimulated PGI2 synthesis in normal and diabetic rat aorta. The serum activity in diabetic rats was less potent than in normal rats. Melittin and dipyridamole were effective in stimulating PGI2 release from diabetic rat aorta. These results suggest that enzymes in the aorta involved in PGI2 synthesis from released arachidonic acid are susceptible to self-inactivation in diabetic rats during the metabolism of arachidonic acid. This may contribute to the defect of PGI2 synthesis in diabetic rat aorta in addition to the decreased availability of the substrate.

Clinical and Light Microscopic Findings on Process of Experimental Periodontitis in Beagle Dogs.

実験的歯周炎成立過程における臨床的および光顕所見の変化について検討した。ビーグル犬7頭の切歯部に, 歯肉溝内綿糸結紮とソフトダイエット飼育によりプラークの蓄積を促し, 結紮後48週までについて経時的に臨床的およびX線所見について評価し, 通法に従って作製した脱灰標本を光顕により観察した。その結果, 歯肉炎症症状は, 実験全期間を通じて高度みられ, X線像における骨吸収は結紮後4週に始まり, 結紮後48週には歯根長の1/2～2/3に達していた。光顕所見では, 結紮後4および6週時に歯周ポケットが形成され, 上皮下結合組織には結紮後の実験全期間を通じて高度の慢性炎症細胞浸潤がみられ, 48週時の骨吸収は, X線像と同様に歯根長の1/2～2/3に達していた。本研究から, 非交換式の歯肉溝内綿糸結紮法とソフトダイエット飼育によりヒト類似の歯周炎が惹起されることが明らかとなった.

Ultrastructure of Cementogenesis at Dentin Surfaces Treated with Hydrochloric Acid.

本研究では, 塩酸脱灰象牙質基質に対する新生セメント質形成過程の超微形態について検討した。1歳ビーグル犬2頭の小臼歯とその頬側歯周組織に対してフラップ手術を行った。裸出象牙質面を骨削除とセメント質除去により形成し, その根尖側半分に0.3N塩酸を5分間塗布し, 術後8週までの透過型電顕試料を作製して観察した。1. 非脱灰象牙質面では, 術後4週に破歯細胞による吸収がみられ, 8週までに新生されたセメント質基質線維と露出した象牙質基質線維とが互いに絡み合っていた。2. 塩酸脱灰象牙質面では, 2および4週時に脱灰象牙質面上に電子密度の高い層状構造を介して新生セメント質基質が形成され, 両基質線維は互いに絡み合っていた。本研究結果から, 塩酸脱灰象牙質基質には直接新生セメント質が形成されることが示された。

Histological Study of Long Junctional Epithelium. Interaction of Epithelial and Connective Tissue Reattachment following Flap Surgery.

歯周外科治療後の歯根面に生じる長い付着上皮と結合組織性再付着の関係について組織学的に検討した。実験には, 1～2歳のビーグル犬4頭の小臼歯とその頬側歯周組織を用いた。Widman改良法と歯槽骨削除により裸出させた歯根のセメント質を完全に除去した。これらの歯根を2群にわけ, 一方には骨遮断群として骨面をポリカーボネートメンブレンで被い, 他方にはメンブレン未処置の非遮断群とし, 歯肉弁を復位して縫合した。処置後8週経過時までの光顕標本を作製して観察した。その結果,1. 骨遮断群では, 4～8週で裸出象牙質面のほぼ全域を再生付着上皮が被い, 新生セメント質の形成はほとんどみられなかった。2. 非遮断群では, 4週で骨遮断群と同様に裸出象牙質面に長い再生付着上皮が接していたが, その根尖側部には新生セメント質が形成され, 8週には再生上皮の歯冠側移動に伴って新生セメント質が歯冠側に伸展していた。

Histological Study of Long Junctional Epithelium. Ultrastructural Findings of Epithelial Attachment after Plaque Accumulation.

実験的に形成したlong junctional epithelium (LJE) におけるプラークコントロール中止後の変化を超微形態学的に検討した。実験的LJEは, ビーグル犬2頭の小臼歯およびその頬側歯周組織に対して歯肉切開・剥離と歯槽骨削除を行い, 裸出歯根面のラミニン (1mg/ml, EYラボラトリーズ社) 塗布法と歯根周囲の裸出骨面をポリカーボネートメンブレン (孔径3μm, ヌクレポア社) で被う骨遮断法を併用し, 術後4週間経過することにより形成した。プラーク付着実験では, LJEを形成した実験群とLJEを形成しない対照群に対して, ソフトダイエットでの飼育とブラッシングの中止によりプラークの付着を促し, 4週までの変化を電顕的に観察した。その結果, 実験群では, プラークコントロール中止後, 対照群と同様に1～4週でLJE細胞間に裂隙が生じて, 次第に深くなり初期ポケットが形成された。超微構造では実験期間を通じて上皮付着構造に変化はなかったが, 2～4週の歯冠側部のLJEには細胞剥離や変性像および細胞間隙の拡大と好中球浸潤がみられたが, 根尖側部のLJEにはこれらの変化は及んでいなかった。

Stimulation by carrageenan of arachidonate 12-lipoxygenase activity in dog gingival tissue.

At 4 h after injection of carrageenan into the gingiva, the 12-lipoxygenase activity of the gingival homogenate was markedly increased. Activity in the cytosol and microsomal fractions was markedly increased when assessed as the specific activity based on nmol/min/mg of protein, and in the cytosol fraction as the percentage distribution of total activity. The 12-lipoxygenase activity in the homogenate from carrageenan-treated gingiva was not affected by either EDTA or calcium ion, or a combination of the two. 12-lipoxygenase activity in both carrageenan-treated and untreated gingiva was inhibited dose-dependently by AA861, a striking difference from its effect on platelet 12-lipoxygenase. There was a marked increase of 12-lipoxygenase activity in experimentally inflamed gingiva compared to the non-inflamed gingiva.