Biochemical data generally agree that the organic matrix in enamel is made up mostly of proteins. No agreement has been reached, however, regarding the structure of the organic matrix, its relationship to the enamel crystals, and its role in the mineralization of the enamel. It has been argued that the organic matrix precedes and guides the formation and orientation of the crystals. Conversely, others have described the matrix as a gel; and evidence has been presented to suggest that the organic structures seen in decalcified sections represent, not preformed macromolecular structures, but organic material adsorbed to the surface of enamel crystals.
A close relationship between organic matrix and the crystal surface exists from an early stage in the appearance of the first, thin, ribbon-like crystal through all subsequent stages of crystal growth. This is indicated by the finding that, during the maturation stage, organic-matrix structures change parallel with changes of crystal size and shape.
Materials identified as amelogenins by their soluble properties make up a relatively small part of the organic materials attached to newly formed enamel crystals. In addition, the amelogenins appear to be diffusely distributed among growing crystals. Materials identified as enamelins, and assumed to account for a major part of crystal-attached material, resist prolonged guanidine-hydrochloride extraction and are tightly bound to the crystal. Extracted from enamel and purified, these two protein groups show a high affinity to enamel crystals in vitro.

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Quantification of water insoluble glucan forming glucosyltransferase (WIG-GTase) in the culture supernatant of Streptococcus mutans (S. mutans) and the effects of culture conditions on WIG-GTase production production were examined. A S. mutans strain producing the greatest amount of extracellular WIG-GTase was selected from 14 references and freshly isolated 70 strains. Tested strains were incubated under aerobic condition in a partially defined medium M4. S. mutans B-13N displayed the strongest activity when WIG-GTase activities of their cultures were determined turbidimetrically. The culture supernatant of strain B-13N had a WIG-GTase activity of 0.72 IU/ml which corresponded to a specific activity of 9.2 IU/mg protein. The increase of turbidity was 0.09 per minute at 340 nm in a standard reaction mixture containing 200μl of the culture supernatant and proportional to the culture supernatant volume in the range of at least 50 to 200μl. Using the culture supernatant directly, quantitative, rapid measurement of WIG-GTase in a culture was accomplished by combining strain B-13N and the turbidimetric assay. The effects of various factors on WIG-GTase production were examined through this method. Strain B-13N cells produced extracellular WIG-GTase activity only during logarithmic growth and the cell-associated WIG-GTase activity was less than 1% of the extracellular enzyme. Enzyme production was affected by temperature the optimum was 33°C, but not by pH in the range of 5.5 to 7.0. When glucose, fructose, or mannitol was the carbon source in a partially defined medium, cells growing on fructose produced more WIG-GTase than cells growing on glucose or mannitol. Cells growing on glucose or fructose produced more WIG-GTase under anaerobic rather than aerobic condition. Enzyme production was liable to be stimulated by increasing Tween 80 concentration, but was not affected by either glucose or (NH₄) ₂SO₄ concentration in a normal growth condition. This experimental system will be useful for examing the inhibitors or stimulators of WIG-GTase production as well as the effects of culture conditions on WIG-GTase production and furthermore, for clarifying the mechanisms of enzyme production and secretion of S. mutans.

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Fibronectin (FN) is a major glycoprotein in vertebrates that is found as a soluble faction in plasma at about 30mg/dl and is widely distributed as an insoluble component of various tissues, in close association with fibrillar structures of connective tissue matrices, basement membranes and certain mesenchymal cells such as fibroblasts. The present study was undertaken to examine the presence and distribution of FN in the periodontal tissues of mice by immunofluorescence using an anti-mouse plasma FN antibody. FN was purified from mouse and human plasma by affinity chromatography on a gelatin-Sepharose 4B. The purified FN gave a single band on SDS polyacrylamide gel electrophoresis and displayed biologic activity such as induction of adherence and spreading of fibroblast and macrophage on the plastic surface at 37°C in a serum-free medium. Antisera of mouse and human FN, which were obtained by hyperimmunization of rabbits with the respective FN, gave a single precipitation line against purified FN and plasma in Ouchterlony's double diffusion. The specificity of these anti-FN antibodies was also ascertained by immunofluorescence staining of FN in fibroblasts and peritoneal macrophages. By immunofluorescence using this specific mouse FN antibody, it was found that FN was distributed in the lamina propria, periodontal ligament and tooth pulp and in the osteocytes of alveolar bone. In aged mice, FN in the periodontal ligament was distributed at a high concentration on the border of cementum, making a clear band.

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Odontoblasts with pure and considerable cell population were harvested from developing bovine mandibular incisors. It was confirmed by light and scanning electron microscopic observation that odontoblasts remained as a layer on the surface of developing dentin when the dental pulp was removed. The cells were obtained as a saline suspension by scraping off from the dentin surface with a microspatual.
The alkaline phosphatase activity in this odontoblast preparation was determined and compared with the activity in other pulp cells.

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In order to determine whether the effect of glucocorticoids (GC) on collagen synthesis in palatal shelf during palatogenesis is responsible for cleft palate induction, morphological and biochemical studies were carried out on mouse fetuses of the steroid-sensitive CF1 strain. Thespecimens used were palatal shelves of fetuses at 11-15 days of gestation treated with or withouttriamcinolone acetonide (TAC) in vivo during days 11-14.
SEM and TEM observations revealed the accumulation of fibrous materials in the intercelluar space of palatal shelves with increase of gestational age. Incubation of the palatal shelves with col-lagenase demonstrated that these fibrous materials consisted of collagen. Quantitative analysis of the fiber density in the shelves from fetuses treated with or without TAC suggested that TACinhibited the formation of collagen fibers in the shelves. The percentage decrease of collagen synthesis in TAC-treated fetuses was greater than that of non-collagen protein synthesis at all gestational ages, indicating a selective inhibition of TAC on collagen synthesis. The inhibition was greatest at day 11.5 which had been reported to be the most sensitive time for administration of GC for cleftpalate induction.
The results obtained in the present study suggest that inhibition of collagen synthesis by GC is involved in cleft palate induction by GC.

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The size distribution of HeLa and KB cells were studied using a particle size distribution analyzer to clarify the threshold of cell size. The distribution of HeLa cell particles ranged from maximum 3050.0 μ³to minimum 600.0μ³, mean value 1855.1μ³, peak value 1550.0μ³and KB cells from maximum 3900.0μ³to minimum 700.0μ³, mean value 2056.6μ³and peak value 1400.0μ³. In conclusion, the results obtained were as follows; 1) The threshold should be determined to the minimum point which the size distribution curve revealed one peak. 2) As the cell suspension medium was kept over 5 minutes, the cell count number decreased due to precipitation. Therefore cell counts should be done with in 5 minutes. 3) As errors in the cell count increased when the cell number in the medium decreased below 4000cells/ml, the cell number in the medium should be kept at over 4000 cells/ml in the cell count.

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Recently, biomaterials are being widely used in the clinical field due to progressin their development. In toxic substances of biomaterials (the tissue culture) is profitable parts, and measurement of cell count is necessary. For measurement of cell count, the electric cell counter has been used in our laboratory.
The basic study on measurement of cell count was performed with automatic blood cell counter with a particle size distribution analyzer. The materials were L-cells and V₇₉-cells subcultured within 4-days.
The results obtained are as follows; 1) The distribution of L-cell particles ranged from maximum 3200μ³ to minimum 350μ³, mean value 1729.1μ³, and peak value 1000μ³. However, V₇₉-cell particles ranged from maximum 2700μ³to minimum 200μ³, mean value 1365.3μ³ and peak value 550μ³. 2) As the cell suspension medium was kept over one minute, cell count numbers decreased due to cell precipitation. Therefore, the cell count should be done within one minute. 3) Error in the cell count increased when the cell number in the medium decreased below 2.0×10³ cells/ml in the L-cell and 2.0×10⁴ cells/ml in the V₇₉-cell.

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Electron microscopic observation was undertaken on four bacteriophages, specifically for Veillonella parvula ATCC17743. It was revealed that the group A phage (N1, N4, N6 and N10) had a hexagonal head of 65-67nm diameter and a wedge-shaped tail of 18nm phage length and 9-18nm width at the base. These phages were classified as type C according to Bradley's definition.
The N1 phage made to be adsorbed to Veillonella parvula ATCC17743 was seen to use negative staining method. It suggested that the receptor was distributed all over the surface of the cells.
By the ultra thin section method, Veillonella parvula ATCC17743 was found to constitute an outer layer, middle layer and plasma membrane, and then it was supported that each layer consisted of two electron dense layers and one less dense layer. Furthermore, when the N6 phage was adsorbed by the host strain, an image suggesting the possibility of the tail length of N6 phage being extended was obtained.

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Free and matrix-bound phosphoprotein (phosphophoryn) fractions were extracted from the bovine incisor dentin, and purified by DEAE-cellulose, SP-Sephadex and gel filtration chromatography. Both components were eluted in broad peaks, when chromatographed on gel filtration column even in dissociative conditions, indicating the predominance of aggregative tendency of phosphophoryn. On SDS-polyacrylamide gel electrophoresis, both components migrated in sharp bands. However, the apparent molecular weights, estimated from the mobilities of standard globular proteins, varied depending on the gel concentration. This non-ideal behavior of phosphophoryn might be due to the effect of its polyanionic structure.
Matrix-bound phosphophoryn is thought to be a tight complex of phosphophoryn and collagen peptides. This complex was different from simple non-covalent association products of these components. Both free and matrix-bound phosphophoryns contained lysinoalanine. But this amino acid could not be a candidate for the cross-link between phosphophoryn and collagen.
The possible role of phosphophoryn in calcification of dentin was discussed.

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This study was aimed at investigating the morphological changes during embryogenesis and the innervation of rat palatal muscles. Wistar rats aged 14.5 days of gestation to 3.5 days after birth were fixed in Bouin's fluid and embedded in paraffin. For the usual microscopy, serial sections were cut, and some of them were used to reconstruct two-dimensionally for examination of muscle distribution. For investigation of the innervation of palatal muscles, the sections were stained by silver impregnation method.
The palatal muscles of rats consist of m. tensor veli palatini (TVP), m. levator veli palatini (LVP) and m. plalatopharyngeus (PP). The TVP appeared initially of three muscles adjacent medially to m. pterygoideus med. at 15.0 days and developed with fan-shaped distribution in the lateral region to the proc. pterygoideus. The LVP appeared as a very small group of myoblasts just below the tuba auditiva and separated from the myoblasts mass of the pharynx. It developed ventrally and laterally, but remained a small cell mass. The PP, initially mingled with the myoblasts mass of the pharynx, appeared distinctly at 17.0 days, then developed rostrally and caudally to form a beltlike shape. Its final shape was observed to be a sphincter at the pharyngeal isthmus. The TVP was obviously supplied with the branches derived from n. mandibularis. The LVP was supplied with three source of nerve fibers: the first was the fibers from the plexus pharyngeus which also supplied the PP, the second was that from the nn. palatini minores, and the last, from a direct branch of the n. canalis pterygoidei.
Subsequently, in order to examine the distribution of motor nerve cells that innervate the palatal muscles, 30 % HRP (Horseradish peroxidase) was injected into the soft palate of the adult rats. HRPlabelled cells were found in the ventrolateral and lateral part of the nucl. motorius n. trigemini, the dorsal part of the middle region of the nucl. n. facialis and in the nucl. ambiguus. These findings suggest that some of the motor nerve fibers to the LVP were derived from the n. facialis and they passed through the n. petrosus major.

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A multi-unit microelectrode recording was performed in the somatosensory cortex SI and its receptive field was defined by light mechanical stimulation to the oral structures such as gingiva, periodontal tissues, palate, tongue and lip of the cat. The oral projection area was found somatotopically at the circumscribed area (about 3×3mm²) in the anterior coronal gyrus and situated more rostrally than the so-called face area. It was found that the area was subdivided into small loci which were characterized by projection from a particular part of the oral structures. The loci examined were represented in order of contralateral lip, contralateral, medial and ipsilateral part of gingiva and periodontal tissues, palate and tongue, in a caudal-rostral direction. Details of the geometric arrangement were as follows; they were distinguished functionally in two parts of the caudal site (contralateral) and the rostral site (ipsilateral) by mid-line (X) and also divided into the medial site (mandibular) and lateral site (maxillary) with an approximate parallel line (Y) to the coronal sulcus. The intersecting angle (φ) between the coronal sulcus and line X/ and the crossangle (θ) between line X and line Y were measured as an index of the representing distortion. Distance (Z) was also measured from the intersecting point of line X and the coronal sulcus to the cross point of the extended line of the cruciate sulcus and the coronal sulcus. The mean values of 10 adult cats were ø=99±24.6°, θ=105±18.6° and Z=4.3±1.4mm. The area mentioned above was also studied cytoarchitectonically in 5 cats. It was confirmed that localization of the loci was defined in area 3b.

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Because macrophages possess an ability of bone resorption and increase remarkably in gingival tissues with periodontitis, bone resorption on the part of macrophages was examined by using macrophages induced in the peritoneal cavity by bacterial cells and peptone. After administration of powders, such as long bone, alveolar bone, human enamel and synthetic hydroxyapatite, to the culture system of macrophages, dissolution of the contents in the hard tissues was examined by measuring the Ca release in the culture media. In the present study, Ca release depended upon the concentration of macrophages. However, no difference in ability of Ca release was found between macrophages induced by peptone and macrophages induced by killed cells of Actinomyces viscosus ATCC 15987 strain. Ca release from hard tissues, each of which was comparatively investigated, was found to be equal in amount between the above-stated two kinds of macrophages. Therefore, it was clear, in spite of the different macrophage-inducing methods, that macrophages possess an ability to dissolve almost all hard tissues in organisms.

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Artificial caries like lesions produced by exposure to the demineralizing fluid were exposed to acousto-optically O-switched Nd: YAG laser. Scanning electron microscopic observations of the acid-etched surfaces of the lased lesions showed reduced acid solubility of both the surface and subsurface enamel.
After the lased lesions were exposed to the demineralizing fluid for 14 days, the acid resistence of these lesions was mainly histologically examined with microradiography, scanning electron microscopy and polarizing light microscopy. Microradiographs showed the appearance of radidensity in the subsurface layer and a small amount of subsurface demineralization. Moreover, a radiodense lamination was shown in the subsurface layer and it showed isotrophy by polarizing light microscopy. Scanning electron microscopic observations of the surfaces and the cut surfaces of the lased lesionsshowed remarkable acid resistance. An atomic absorption analysis indicated the low value of Ca density inthe demineralizing fluid. Ca linear density in the cut surfaces of the lesions showed stable intensity at the subsurface layer by X-ray microanalyser. In the timely examination of demineralization, radiodensity with lamination appeared in the early stage of demineralization.
Microradiographs of the lased lesions exposed to the remineralizing fluid for 7 days showed marked radiodensity in the subsurface layer and this indicated that laser irradiation to the artificial caries like lesions may accelerate remineralization.
These results suggest that acousto-optically O-switched Nd: YAG laser irradiationmay be effective for prevention of development of dental caries.

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Na⁺, K⁺-dependent ATPase was purified from porcine kidney by the modified method of Lane et al.
A) Binding of ouabain to Na⁺, K⁺-dependent ATPase during the reaction.
The amounts of a phosphorylated intermediate (EP) and ouabain bound to the enzyme during the ATPase reaction were measured in 2.1 mM MgCl₂ and various concentrations of NaCl and KCl at pH 7.5 and 20°C. In the presence of NaCl and the absence of KCl, the molar ratio of EP and bound ouabain was 1:2. In the presence of NaCl and KCl, the ratio was 1:1. In the both cases, the amount of bound ouabain was equal to that of EP in the absence of ouabain. These findings suggest that the functional unit of the transport ATPase is a dimer.
B) Effect of digitonin treatment on the ATPase. Winter et al. reported that the hypothesis that digitonin dissociated the Na⁺, K⁺-dependent ATPase into αβ monomers appeared to be supported by two independent types of measurement: the change in crosslinking susceptibilities and the hydrodynamic properties of the digitonin-generated products. In the present study, the amount of EP and phospholipid, p-NPPase activities and the ATPase activities of the digitonin-treated enzyme were measured.
The following results were obtained.
1) The amount of EP was equal to that of the control.
2) The rate of K⁺-dependent EP decomposition was decreased to about 5 % of the control level by treatment with digitonin.
3) The apparent affinities of the ATPase for ATP, Na⁺, Mg²⁺, and K⁺ were changed by treatment with digitonin. The apparent high affinity of the ATPase for K⁺ was decreased to about 5% of the control level by treatment with digitonin.
4) The amount of phospholipid in the ATPase was reduced to about 65% of the control value by treatment with digitonin.

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No report among studies made to date on the split-line have dealt with bone graft. Therefore, a portion of a rib was resected from mongrel dogs, one to two years old, and autogenous bone transplantation on the mandible was performed. In the transplantation, two methods were used: the split-line direction oriented to or at right angle to that of the original mandible. Observations of the split-lines at the graft site were carried out for five months beginning two weeks after the grafting. The results were as follows: 1. All of the original split-line pattern in the transplant disappeared and irregular split-lines appeared instead at the site. 2. In comparison of the cases of the split-line direction of the transplant oriented to that of the original mandible bone with those of the direction at right angle, reorganization of the pattern of the irregular split-lines appeared earlier and the pattern tended to become oriented to that of the original mandible bone in the former. On the basis of the observation findings, it is postulated that all of the fiber structure can be remodeled and reorganized. It is also considered that in grafting, orientation of the split-line direction to that of the original mandible bone will expedite the healing process.

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The present study was designed to clarify the relationship between the architecture of the alveolar bone and its neighboring bones and masticatory stress. The dogs whose lower premolars and molars on one side were extracted were kept for 64-181 days. During the experimental period, the animals were injected with tetracycline and calcein continuously as shown in Fig.1. The large ground sections showing the frontal section of the maxillary bone and its neighboring bones (Fig.2) were first microradiographed and, then, were subjected to fluorescence microscopy. From a montage of the fluorescence micrograms, a colour tracing of the labelling sites (tetracycline- red, calcein-green) was made on a plastic sheet. The two images obtained by these different methods from the same ground sections were compared precisely.
Soon after the antagonists were extracted, the alveolar bone proper along the periodontal spaces of the upper premolars and molars begin to become narrower through progressive resorption from the side of the spongy area of supporting bone, although lamellar apposition of bone progresses at the side of the periodontal membrane. On the other hand, the bone trabeculae in the spongy area of the supporting bone becomes thinner and shorter gradually. This is probably due to retardation of apposition and acceleration of resorption of the bone. The compact bone at the periphery of the maxillary bone also decreases in width very gradually.
About three months after the antagonists were extracted, the changes in architecture and remodelling pattern become observable in the zygomatic arch, frontal bone and perpendicular plate of the palatine bone, but are not observed in the horizontal plate of the palatine bone even after six months.

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It has been reported that macrophages in the focus of periodontitis participate in the destruction of the periodontal conective tissues and resorption of the alveolar bone. It can be supposed that clarification of the ability and mechanism of bone resorption by macrophages offer a very important clue to the search for prevention of periodontal diseases. Therefore, the solubility of various hard tissues by mouse macrophages was observed in vitro.
Rat long bone that has the crystalinity of apatite, rat alveolar bone, human enamel and synthetic hydroxyapatite were selected as hard tissue samples. The solubility of these samples was compared and the effect of ash treatment was examined.
Solubility of the samples was determined by the atomic absorption method measuring the calcium dissolved in the macrophage culture media.
It was seen that the hard tissues samples were dissolved by macrophages with or without ash treatment. The solubility was found to differ between samples but the solubility did not always correspond to crystalinity of the samples. By ash treatment, the solubility of the samples was in creased.

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Existence of bacterial enzymes (chondroitinase ABC and hyaluronidase) in the dental plaque obtained from 50 volunteers (22 years old) and clinical health demonstrated by fluorescence antibody technique.
Chondroitinase ABC was detected in the plaque of all volunteers and hyaluronidase was demonstrated in 44 of the 50 volunteers. Fluorescence of chondroitinase ABC and hyaluronidase was observed as a diffuse, granular or fibrillar pattern in the plaque. From these fluorescence patterns, it was indicated that the diffuse pattern represented the exudate from certain bacteria, and granular or fibrillar pattern showed localizationin in the cytoplasm of the bacteria. It was also suggested that the bacteria which produced chondroitinase ABC and hyaluronidase were a type of cocci or fusiforms.

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The ultrastructural changes in the process of extra and intracellular degradation of collagen fibrils and fibroblasts in the periodontal membrane of rat mandibular molar after discontinuation of occlusal function were investigated with an electronmicroscope and histochemically for demostration of the activity of acid phosphatase (Barka and Anderson method). In addition, histological changes in the fibrous structure of the salt-insoluble collagen. The following results were obtained. 1) After discontinuation of occlusal function, the salt-insoluble collagen fiber bundles of the periodontal membrane underwent more rapid degradation on the alveolar bone side than on the cementum side. Acid phosphatase activity became much more increased in the fibroblasts on the alveolar bone side than on the cementum side of the periodontal membrane. 2) Electron microscopically, collagen fiber bundles mostly disappeared and became dispersed on the alveolar bone side. However, dispersed individual collagen fibrils usuallay exhibited banding structures. 3) In these areas, many of the fibroblasts phagocytosed small number of collagen fibrils. Some of collagen contanining vesicles showed the activity of acid phosphatase and indicated development of phagolysosomes. 4) Fibroblasts with collagen containing vescles seemed to be more increased on the alveolar bone side than on the cementum side. 5) The present study revealed that diecontination of occlusal function results in increased activity of collagen degradation of the periodontal fibroblasts and normal occlusal force may inhibit the activity. 6) Local injection of cathepsin D induced rapid marked degradation of extracellular collagen fibers. At the same time, fibroblasts showed multiple phagocytosis of collagen fibrils in newly developed numerous cytoplasmic process and cytoplasma.

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