Possible roles of calcifying matrix proteoglycans (PG) in a chain reactions of calcifying process were discussed from a view point of the unique physico-chemical properties of PG.
PG, as a diffuse polyelectrolyte macromolecule, networks with collagen fibres and holds the positions of matrix vesicles (MV) and a space for hydroxyapatite crystals. This gelatinous networks supply microchannels of Ca and phosphoproteins (PP) from cell to calcifying front. This transportation may be promoted by exclusion volume effect and negative charges of the gel.
MV at the calcifying front are punctured by growing apatite seeds and then release a protease associated with MV. This protease decomposes the PG interfering crystal growth.
Decomposed PG, dephosphorylated PP, water etc. are excluded and back-flowed from the calcifying front.

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The purpose of this investigation was to study the localization of bacterial chondroitinase ABC in the human gingival tissue with periodontal disease and influence of the periodontal tissue due to the enzyme by use of a fluorescence antibody technique and histopathological examination.
The presence of chondroitinase ABC in the inflamed human gingivae was demonstrated in all 27 subjects. The existence was mainly in the subepithelial connective tissue. Intensive inflammatory cells, hyaline degeneration and osteoclastic bone resorption were recognized in the rat periodontal tissue injected with bacterial chondroitinase ABC.
From the above histopathological findings 7 it was suggested that osteoclastic bone resorption was brought about by the degradation of bone matrix formed by osteoblasts in the progress of physiolocal bone remodelling.

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The cell population kinetics of buccal and palatal mucosal epithelium of C₃Hf/He mice were investigated by the autoradiographic method following a single intraperitoneal injection of ³H-thymidine. ³H-thymidine was injected to the animals at 22:00-23:00, when a high rate of labelling index was obtained because of its diurnal change. The experimental mice were sacrificed at various times after labelling and used for microscopic observations. The cell cycle time was determined by PLM analysis, developed by Takahashi & Mendelsohn (1971).
The results indicated that the cell cycle time of the basal cell was 43.8 hrs., the buccal mucosal epithelium and 55.5 hrs. the palatal mucosal epithelium. The results of the analysis of NB/NT (Brown & Oliver) showed that about 80% of the labelled basal cells in both mucosa migrated to the superficial cell layer. However the pattern of basal cell migration of buccal mucosa was different from that of palatal mucosa, i. e. migration in the buccal mucosa was continuous and in palatal mucosa intermittent. The turnover time of cells estimated by two the different methods respectively, that is, Brown & Oliver (analysis of NB/NT) and the method estimated from the peak time of the labelling indices in the basal layer and the granular layer. The results indicated that the turnover time of epithelial cells in the buccal mucosa was shorter than in the palatal mucosa.

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Prostaglandins have been recognized recently as one of the important chemical mediators in periodontal disease. The present study is aimed at studying the metabolic alteration in periodontal tissues mediated by prostaglandins.
The effect of prostaglandin E₁ (5 to 200ng/ml) on bovine periodontal ligament cells was investigated by using the short term incubation system with succinate-1, 4-¹⁴C as the radioactive tracer. The extracellular medium was analysed for TCA cycle intermediates and synthesized macromolecules.
Organic acid analysis showed that the synthetic ratios of the pyruvate: malate, and citrate:α ketoglutarate were altered by PGE₁. Especially, α-ketoglutarate: isocitrate ratio increased markedly with 5ng PGE₁/ml.
Macromolecular sythesis was inhibited at low concentration and accelerated at high concentration of PGE₁. The products over 65 K dalton decreased about 1/3 with 5ng PGE₁/ml, and increased with 200ng PGE₁/ml, but the substances of 32-65 K dalton increased about 1/4 with 5ng PGE₁/ml and decreased 15.6% with 200ng PGE₁/ml.
It was found that the synthesized macromolecules of 59 K dalton estimated on the slab SDS polyacrylamide gel electrophoresis was remarkably induced by the low concentration of PGE1 (5ng/ml).
These results demonstrated that PGE₁ is one of the mediators of periodontal tissue cells alterated their organic acid metabolism and macromolecular synthesis.

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In order to investigate the growth mechanisms of hydroxyapatite (HA) in dilute solutions, it is very important to obtain information about the growth rate and the degree of supersaturation with respect to HA. The aim of the present study was to discuss how one can obtain noise-less growth rates based on experimental kinetic data.
Precipitation kinetics on seeded HA crystals at 37° were highly reproducible in the system used. The precipitation rates were determined here in three different ways:(1) fittings of the experimental growth curves to a polynomial, (2) approximation of the growth rate by an empirical parabolic rate law, and (3) the so-called graphic approach, taking an averaged change in solute concentrations between small time intervals.
For the last approach, numerical values of PO₄ every min were calculated from the pH data as well as initial solution compositions. Careful examination of the polynomial and parabolic approaches revealed that small but non-negligible differences occur between the experimental data and the calculated growth curves, especially in the early stage of the precipitation taking place upon addition of seed crystals. One can conclude that the graphic approach is most adequate for describing the dependence of the growth rate upon the degree of supersaturation, although one should further modify this method in order to reduce scatterings involved in the calculated numerical values.

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The degree of supersaturation (DS) with respect to calcium phosphates can be expressed in terms of ionic activity product/thermodynamic solubility product of each of the salts concerned.
In this report, we first described that concentrations of ionic species in the solutions are calculated from mass balance, electroneutrality, proton dissociation and calcium phosphate ion-pair association constants by successive approximation of the ionic strength, and that the activity coefficients are calculated from the Debye-Hückel equation.
The kinetics of growth of hydroxyapatite (HA) seeds was studied at 37°C over a wide range of initial supersaturation with respect to HA. The results clearly showed that there are functional relationships between the initial precipitation rate, R_o, and the initial DS, empirically given by R₀=K (DS-1)ⁿ, 1≤n≤2, where K is the rate constant. The precipitation kinetics obtained under high DS (IP/K_s (HA) =10¹²) deviated from these functional relationships, most possibly due to involve an intermediate phase during crystal growth of HA.
It was also confirmed that the initial precipitation kinetics was affected in the presence of NaCl and KCl, which were added as background electrolytes. An important finding was that the reaction time courses on the basis of DS vs time, can be reproduced under the same DS conditions, even if the initial compositions of the solutions were different. These results provide further evidence that the driving force of HA crystal growth is the degree of supersaturation.

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Vitamin (Vit) C and Vit K influence immune and inflammatory response and are reported to regulate neutrophil metabolism and functions. In this study, in order to investigate the direct effect of Vit C and Vit K on macrophage migration, in vitro chemotaxis assay was performed by the membrane filter method using guinea pig macrophages.
Glycogen-induced peritoneal macrophages were placed on the upper wells of chemotaxis chambers, which were separated from 10^-8M N-formylmethionyl-leucil-phenylalanine (FMLP) as chemoattractant by 5-μm pore size polycarbonate filters. Vitamins were applied to the lower wells and the upper wells at the same concentrations. The chambers were incubated at 37°C for 90 min and the numbers of macrophages which migrated through the filters were quantitated.
The effect of Vit C on macrophage migration was determined with L-ascorbic acid and sodium ascorbate. L-Ascorbic acid (5×10^-5M to 10^-3M) or sodium ascorbate (5×10^-5M to 5×10^-3M) did not extend the effect on the macrophage migration to FMLP and to buffer control, whereas 5×10^-3ML-ascorbic acid suppressed the chemotaxis to FMLP.
Vit K₃ and K₅ had no effect on the migration to buffer control at the concentrations of 5×10^-7M to 10^-5M. On macrophage chemotactic response which was induced by 10^-8M FMLP, 5×10^-7M to 5×10^-5M concentrations of Vit K₃ caused a dose dependent suppression. In case of incubation with Vit K₅, the macrophage chemotaxis was not affected at 5×10^-7M to 10^-5M concentrations but were completely inhibited at 5×10^-5M.

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The postnatal differentiation of granular intercalated duct cells in the mouse submandibular gland and the effects of estrogen were examined by light and electron microscopy using the zinciodide osmium (ZIO) staining technique.
At prepuberty, only the terminal tubule cells exhibited ZIO-positive, whereas the acinar cell, the proacinar cell and the non-granular intercalated duct cell were ZIO-negative. As to the ZIO staining properties of terminal tubule cells, the three types could be distinguished, Type I: the whole cytoplasm including nucleus was positive, Type II: the rough endoplasmic reticulum and Golgi complex were positive, and Type III: only the Golgi-associated vesicles were positive. Type I was identified with the cell which contained many polysomes and a few secretory granules. This observation confirms that ZIO staining technique exhibits the ability of granular synthesis. The frequency of ZIO-positive cells was high during the first week of life and greatest decrease was observed a little before weaning. A drastic change in cellular composition of the intercalated duct occured following weaning. Between 3 and 4 weeks of age, the terminal tubule cells disappeared in the male. At 28 days of age, ZIO-positive cells were absent in the male submandibular gland but granular intercalated duct cells were ZIO-positive in the female.
When male and female mice were treated with estradiol-17β, the non-granular intercalated duct cells showed ZIO-positive, although the cells of intact mice were negative. In addition to these facts, we observed granular synthesis in the non-granular intercalated duct cells of the female.
These results suggest that terminal tubule cells and granular intercalated duct cells are converted into non-granular intercalated duct cells. The granular intercalated duct cells in the adult female can be considered as remnants of the terminal tubule cells.

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The present study was undertaken to investigate the correlation between clinical pain and the level of β-endorphin in blood plasma. We measured the concentration of β-endorphin and cortisol in blood plasma of patients with oro-facial pain.
The β-endorphin levels of patients were higher than the levels of healthy volunteers (control group), but it was not significant, whereas, the cortisol levels of patients were significantly higher than control levels. Patients classified as having postoperative pain were found to have significantly higher β-endorphin levels than the control group, but the cortisol levels did not change. The β-endorphin levels in patients with trigeminal neuralgia were lower and the cortisol levels significantly higher than those of the control group. Patients with oro-facial pain were classified into acute and chronic pain group. In the acute pain group, β-endorphin levels were significantly higher than in the control group but the levels in the chronic pain group did not change.
There was a correlation between the clinical pain score and plasma β-endorphin level in the acute pain group but not in the chronic pain group. These results suggest that plasma, β-endorphin increases in case of the transient pain and decreases in case of the continuous pain. Further studies, however, will be necessary to discover whether plasma β-endorphin can play any role in the pain control mechanism.

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The allosteric effects of alkaline phosphatase from calf long bone were investigated at 20 (or 19), 37 and 45°C in 50 mM Tris-HCl buffer of pH 7.4 and 50 mM Na₂CO₃-NaHCO₃ buffer of pH 10.2 or 10.6. The bone alkaline phosphatase exhibited two types of allosterism; one was positive cooperativity in the high alkaline pH region and the other negative cooperativity in the physiological pH region. Negative cooperativity at 37°C was revealed as so-called “half-of-the-sites reactivity” which generally appeared under tight subunit interaction permitting only a high affinity site for full operation.
Based on the negative cooperativity of bone alkaline phosphatase, three possible physiological roles were proposed in relation to calcification process in the hard-tissue system.(1) even under low level of organic phosphates as substrate, the enzyme was able to supply inorganicphosphate not only to phosphate-requiring metabolic systems but to calcification sites.(2) the enzyme was able to supply inorganic phosphate at a constant rate by readily attaining maximal velocity (Vm), thus controlling the overall homeostasis of phosphate metabolism by itself.(3) under circumstances of excess intracellular substrates, caused by membrane transport disorder, for instance, the enzyme was able to immediately hydrolyze them by permitting both active sites to fully operate, thus preventing unphysiological formation of amorphous calcium precipitates in the presence of such excess organic phosphates.

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The inhibition effects of tetramisole, levamisole and R-8231 on the activity of alkaline phosphatase from calf long bone were investigated at 37°C in 50 mM Tris-HCl buffer of pH 7.4. Tetramisole, levamisole and R-8231 exerted approximately 42, 50 and 52% inhibition to partially purified alkaline phosphatase activity, respectively. One of two active sites in the enzyme molecule was a high affinity site, of which kinetic constants (K_mI and V_mI) were demonstrated to be invariable regardless of the presence or absence of these three inhibitors. As to the low affinity site, on the other hand, the K_mII and V_mII values suffered 45-50% and 24-30% decrease, respectively. It was concluded that approximately 50% overall inhibition of enzyme activity by tetramisole, levamisole or R-8231 was specifically attributable to the low affinity site of the enzyme.

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Herpes Simplex Virus type 1 (HSV-1) is frequently associated with oral lesion. Recently, strain differentiation of HSV have been made by cleavage profiles of the HSV-DNA with restriction endonucleases.
Although the two isolated strains are related molecular-epidemiologically from the viewpoint of the cleavage profiles, they are quite often not completely identical. As far as the criteria of HSV-1 strain differentiation is concerned, therefore, the heterogeneities were analyzed among the clonally related HSV-1 stocks and subclonal ones by restriction endonuclease cleavage profiles.
The clonally related stocks were prepared from HSV-1 standard strain (Maclntyre) and isolate (nHS). The subclonally related stocks were similarly prepared from HSV-1 standard strain (HF). The DNA of infected cells were extracted from these stocks. The restriction endonuclease such as Bam HI, Kpn I and Sal I were used for cleavage of the DNA. After cleavage, the fragments of DNA were separated by gel electrophoresis.
The results were as follows:
1) The sizes of many fragments, which were derived from the regions spanning the unique repeat junction and the repeated regions, were heterogeneous.
2) Several fragments such as Bam HI-D, Kpn I-B, J and Sal I-B, derived from the unique regions, were heterogeneous as well.
3) The sizes of the other unique fragments were stable, and the appearance or lack of cleavage sites could not be recognized.
These findings suggest that the regions of the HSV-DNA were variable during replication stage of viral DNA. In order to establish identification of HSV-1 isolates, therefore, the results of this study should be considered.

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In order to clarify the histological characterization in suture, the forming process and morphological changes to external factors in the sagittal suture of the mouse (C57BL/6) were examined chronologically and histologically with light-and scanning electron-microscopy.
In the early developmental stage, the suture formation began after the cells, situated between the right and left parietal bones, were pushed up and down and became a spindle-like condensation. This cellular condensation seemed to play the role of blastema in the suture formation. In the completed sagittal suture, the distance between the bones was 50-80μm. Collagen fiber bundles of 5-10μm in diameter were embedded in the sutural surface and these bundles showed a complication at the center of the suture.
Interdigitation of the superior margin gradually became intense from the anterior to the posterior region, while that in the inferior margin remained entirely poor. Bone remodeling was vigorous on most of the sutural surface but most of the inferior area remained a resting surface.
The sagittal suture thus seemed to act as a buffer zone to the external factors and was quite similar to the articulated structure which remained mobile. In this suture the inferior area seemed to be the pivot for the above movements.
In the restoring process, the new bones were mainly formed at the dura mater but no sutural regeneration was observed. On the other hand, vigrous bone formation was seen at both the dura mater and the sutural surface in the extension and reduction of the sagittal suture. This suture later regenerated. The suture, which was kept at 50-80μm distance between bones, showed poor connection compared with that in normal stages.
Due to these facts, it is necessary to keep the 50-80μm distance between bones in a sagittal suture. It is realized that the dura mater possesses a capacity for intense bone formation.

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Solubility of apatite powder and sinter in acetic acid buffer solution was markedly decreased when treated with acid phosphate fluoride solution followed by lanthanum chloride solution. The fluoride treatment was prepared with calcium fluoride on the surface of the apatite powder and converted to amorphous lanthanum fluoride with lanthanum solution, which were analysed by X-ray diffraction and thermal analysis. The treatment did not discolor the teeth.
From these results, it is suggested that the fluoride-lanthanum treatment could be extended to topical application in clinical use.

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It was previously reported by us that fluoride treatment followed by a lanthanum solution treatment (F-La treatment) to synthetic apatite powder provided higher resistance to acid solution than acidulated phosphate-fluoride treatment (APF treatment). In this paper, the two step treatment has been successfully extended to apatite sinter and human permanent teeth to investigate the possibility of application for dental caries prevention.
The surface of the apatite sinter with the F-La treatment was covered with spherical compounds which consisted of calcium fluoride and amorphous lanthanum fluoride based on SEM analysis. These compounds remained after the acid solubility test for 1 hr. Etching the human permanent teeth with phosphoric acid solution above one mol/l removed sufficient oral organic substances (pellicle), giving a chemically reactive surface. The human permanent teeth subjected with the F-La treatment had about two times higher acidic resistance than with the APF treatment. In the analysis by EPMA, lanthanum and fluorine were present about 15μm in depth from the enamel surface and remained after the acid solubility test for 1 hr.

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In an effort to learn more about succinate-1, 4-¹⁴C metabolism in the connective tissue cell, metabolic alteration in the L-929 fibroblast cell was studied at various culture stages.
Newly-synthesized organic acids measured by chromatographic elution each day were by average: malic acid (22%), fumaric acid (11%), lactic acid (7%), citric acid (5%), pyruvic acid (4%) and substance A (23%). Total organic acid production increased almost 2-fold by the 8 th day of culture. Moreover, the ratio of newly-synthesized macromolecules to those incorporating succinate-1, 4-¹⁴C continued to increase after the cell population became a monolayer at the fourth day.
This study shows that succinate-1, 4-¹⁴C turnover is extensive and rapid with L-929 cell proliferation. As cultured cell sampled are often taken when the monolayer stage is reached, the study inculudes a comparison of biological composition in culture medium with and without cells. Measurement of protein, uronic acid and hydroxyproline showed increases of 26%, 14% and 47%, respectively, in the culture medium with cells.

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Histological examinations of erupted teeth and tooth germs in juvenile and adult dog salmon, Oncorhynchus keta, were carried out by light microscopy, scanning and transmission electron microscopy. As results of these observations, it was found that 1) cap enameloid may only be present in the teeth of juveniles and 2) the inner dental epithelial cells (i. d. e. cells) had great influence on the absorption of the majority of the cap enameloid matrix containing collagen fibers rather than on the formation of tooth matrix.
In the teeth of juveniles, the tips of teeth were recognized as hypermineralized and collagen free portions by light microscopic, microradiographic, scanning and transmission electron microscopic observations. In the teeth of adults, however, no such features were seen in the tips of teeth. It seems that cap enameloid is absent in adult teeth.
In the stage of matrix formation, the features of the inner dental epithelial cells (i. d. e. cells) indicated that the cells had begun their secretory activity. However, the morphological features of i. d. e. cells indicated that activity of the cells in matrix formation was at a low level and that a great quantity of epithelial secretions widely spread out in the tooth matrix was not obtained except around the basal lamina.
On the contrary, active absorptive function at the distal margin of i. d. e. cells is expected and it is considered that absorption of organic substances from the cap enameloid area by i. d. e. cells had already begun in the late stage of matrix formation. Therefore, in the case of juvenile dog salmon, it is assumed that the i. d. e. cells play little part in the formation of tooth matrix and that the majority of the tooth matrix originates from the odontoblasts.
In the stage of maturation, the taller and clear i. d. e. cells possessing a remarkable ruffled border at the distal margin were found in the portion surrounding the cap enameloid. It is assumed that such cells absorb the organic matrix containing collagen fibers from the cap enameloid area and make it a hypermineralized area. On the contrary, though the i. d. e. cells corresponding to collar enameloid became taller at this stage, the remarkable ruffled border was not found in these cells. The outermost layer of the collar enameloid area, about 0.5μm in thickness, became more electron-dense and the crystals, which appeared to be arranged perpendicular to the tooth surface, were gathered densely. Considerable difference in the formation mechanism may be present between cap and collar enameloid.

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Fifty-eight obligately anaerobic rods, which were isolated from human carious dentine and produced lactic acid as a major end product, namely Lactobacillus, Bifidobacterium and Actinomyces, were identified. Of 26 isolates ofLactobacillus, 9 isolates were identified as L. minutus, 8 isolates as L. plantarum, 3 isolates as L. crispatus and 3 isolates as L. catenaforme (including1 L. catenaforme-like strain). One isolate resembled L. cellobiosus and one resembled L. brevis. The other one could not be identified. Of 19 isolates of Bifidobacterium, 8 isolates were identified as B. bifidum (including 3 B. bifidum-like strains) and 11 isolates resembled B. breve. Of 13 isolates of Actinomyces, 7, 5 and 1 isolates were identified A. israelii, A. naeslundii and A. Odontolyticus, respectively. The species identifications of these isolates provide informations that the obligately anaerobic species of genus Lactobacillus and the obligately anaerobic strains of Actinomyces species were isolated frequently from carious dentine with adoption of an anaerobic glove box system.

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Microdissection under SEM was attempted by using a micromanipulator functioning with screws and equipped with a φ 0.1mm tungsten wire as the tip instrument. The specimens in these experiments, were rat embryos immersed in a 1.25% glutaraldehyde (GA)-2% paraformaldehyde (PFA) mixture and some organs from adult rats perfused with 2.5% GA-4% PFA mixture. It was shown that the Bowman's capsule of the kidney corpuscle exposed on the freely fractured surface can be removed by a tungsten probe on the micromanipulator, and to make the gromerulus visible, the pericardium of the rat embryo could be peeled off and the anlage of the heart exposed and dissected further to display the intracardial space. Microdissection of the eye of an embryo was also tried.
It showed the possiblility of freely and arbitrarily making objects visible, objects which until now have been obtained only by chance. This method will have wider applications especially in embryological studies, which depend on the skill of experienced investigators.
The various problems to develop more accurate microdissections were pointed out and discussed. The solutions to some of these problems are suggested.

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Most bones other than the membranous bone are formed by endochondral ossification in the embryonal stage. On the other hand, uncalcified bone matrix gradually apposes to the surface layer of the existing fetal bone formed by endochondral ossification, by osteoblasts of the formative layer of the periosteum and finally the basic form of the bone is completed. Therefore, in order to clarify the surface layer of the fetal bone, i. e., the three dimensional ultrastructure of the formative bone layer, we observed the surface layer of the mandibular body region of the fetus in the late embryonal stage by high resolution scanning electron microscope.

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A simple method for assessing pain and analgesia in mice was developed by modification of the formalin test. Formalin was injected into the forepaw of a mouse and the durations (in sec) spent in licking and biting responses were measured. These behavioral responses being very distinct, pain assessment was easily performed without the experimenter's subjective assessment. The use of the duration of pain during each 5 min block of 2 peak points enables determination of the analgesic effect quantitatively and shortening of the experimental time. As a low concentration of formalin (0.5%) was used in this method, the analgesia of weak nonnarcotic analgesics was detectable as well as that of narcotic ones and tissue damage was not elicited. Furthermore, this method enables to distinguish roughly the action site of analgesics between the central and peripheral one.

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