THE ELECTRON-MICROSCOPIC STUDIES AND ROUGHNESS-TESTING OF NATURAL SURFACE C-AVITAMINOSIS

Transcription

1 236 THE ELECTRON-MICROSCOPIC STUDIES AND ROUGHNESS-TESTING OF NATURAL SURFACE OF TEETH BY EXPERIMENTAL C-AVITAMINOSIS BY ICHIRO SUMr I INTRODUCTORY Despite the fact that vitamin C had been discovered comparatively earlier than other kinds of vitamins, the inquiry into its exact mechanism still leaves much room for future researches. It is well known that deficiency of this vitamin causes various functional disturbances and many findings have been already made public with regard to its effects on teeth and their paradental tissues. It is also recognized that V. C has much to do with the calcification of teeth and it is generally believed that V. C does not directly control the metabo lism of Ca or P which is the chief constituent of teeth, but its deficiency decreases the ferment activity of forming cells thus bringing about the atrophy of amelo blast and odontoblast. Although many reports are published with regard to the natural or pathological surfaces of teeth by means of the electron microscope in recent years, the usual method fails to grasp the condition of the roughness in three-dimensional or numerical terms. In the present study, the author has undertaken a series experiments in which V. C was artificially removed and supplied in order to see the change in the gradual aspects of surface roughness. They have been observed by means of the electron microscope and the roughness-testing was also carried out to render the result in numerical terms. II EXPERIMENTAL METHODS 1) Experimental animals As experimental animals, use was made of 350 guinea pigs with the average weight of g. Prior to the experiment, these animals were fed for a certain period so that they might be more adapted to the experimental environ ments. They were divided into the following three groups. The first group: Control group Laboratory of Clinical Study, Nihon Univ. School of Dentistry (Director: Prof. SETSUZO OKINO)

2 The second group: The third group: 2) Experimental techniques Group of V. C deficiency Group of V. C supply A. Experiments of V. C deficiency and supply i. V. C deficiency experiment Food of the following ingredients have been prepared after Sherman and the experimental animals were fed on it. Bean curd refuse 59 % Skimmed milk 30 % (it was heated for two hours at the completely destroyed) Fresh butter 10 % Salt 1% Cod-liver oil 0.5 cc (it was administered on the weekly basis) Fig Control group 2. C-avitaminosis group 3. V. C supply group As is given by Fig. 1, 10 animals made up a group and these groups were allocated to the six different lengths of time 1 st group (5 days), 2 nd group (7 days), 3 rd group (10 days) 4 th group (2 weeks), 5 th group (3 weeks) and 6 th group (4 weeks). ii. V. C supply experiment When the animals had shown the actual sign of V. C deficiency after two or three weeks, they were subjected to the supply experiment. (a) V. C injection Simultaneously with the feeding of the animals with the V. C deficient food mentioned above, 10 mg of Ascortin (Tanabe Mfg. Co.) was injected daily for 15 weeks consecutively. As is given by Fig. 1, the animals under this experiment were classified into 13 groups with 10 animals in each group 1 st group (5 days), 2 nd (7 days), 3 rd group (10 days), 4 th group (2 weeks), 5 th group (3 weeks), 6 th group (4 weeks), 7 th group (5 weeks), 8 th group (6 weeks), 9 th group (8

3 238 weeks), 10 th group (10 weeks), 11 th group (12 weeks), 12 th group (14 weeks) and 13 th group (15 weeks). (b) of fresh lemon juice via mouth 2 cc of fresh lemon juice was added to the above V. C deficient food twice daily and by this means the recovery of V. C was examined. 13 groups with the same lengths as are with the experiment under (a) above were subjected to this test. c) ( Control group Animals of the control group have been fed on the food of the following ingredients. Bean curd refuse 59 % Skimmed milk 30 % Fresh butter 10 % (it was not heated) Salt 1% Liver oil 0.5 cc (once on the weekly basis) To this were added some fresh vegetables and fruits occasionally. B. Observation of experimental animals i. Change in weight Changes in weight of the experimental animals have been examined with the weight of the control group as standards, both for V. C deficient and supply animals. ii. Growth rate of incisors On the neckline of lower incisors of both the experimental and control animals a shallow notch were marked by means of a file and the distance between the notch and the cutting edge was measured on the three-day basis. iii. Macroscopical observation of enamel surface As against the surface roughness, shade and transparency of the control incisor, those of V. C deficient animals were observed macroscopically. The results were compared with the transition to normalcy in the V. C supply group. iv. Macroscopical observation of paradental tissues As against the general symptoms and paradental tissues of the control animals, those pathological changes in V. C deficient animals were examined. At the same time, the transitional change to normalcy in the paradental tissues of V. C supply animals were observed in the comparative light. v. Electron-microscopic findings Incisors of the experimental as well as control animals were used as chief materials. They were subjected to the electron-microscopic examination. One third portion of the incisor tooth-neck was prepared as the microscopic material. The electron microscope was A-type of HU-10 Hitachi Mfg. Co., with the accelerating voltage of 75 kv. Use was made of films for the purpose of replica available on the commercial market. The film (acetylcellulose) was cut a little larger than the surface of the microscopic material and immersed in the solvent of methyl acetate. It was put on the material as quickly as possible. As the solvent easily evaporated in a few minutes, the shape of the material remained on the film surface after the latter was removed by a pincette. The film was attached to the slide glass by means of cellophane tape and placed in the vaccum evaporator in which the shadowing by

4 239 chromium was done. Following this process, the mesh of 2 mm was drawn on the film by means of a pin. Having been cut to a proper size, it was placed on. another slide glass on which a thin layer of paraffin was painted as protection film. The films thus processed were immersed in the solvent for a couple of minutes. After they were placed in the dessicator, they were heated together floated in the solvent, they were placed into fresh solvent for once or twice. As the final process, they were collected by the sheet-mesh and observed under the magnification of As there were observed certain individual differences in the experimental animals, the standards were taken of those findings which were considered to be mean. Also the mean value in the results of roughness-testing was used as reference. vi. Roughness-testing of natural surface of incisor The natural surfaces of incisor of both the experimental and control animals were subjected to the roughness-testing in the following manner. The findings of macroscopical as well as microscopical observations were used as reference. Fig. 2 (a) Roughness-testing machine. (Fig. 2) In the present roughness-testing, use has been made of Yamamoto point type roughness-testing machine. The mechanism of the machine is given by Fig. b) Testing method Lower incisors of both experimental and control animals were immersed in the H2O2 solution for 24 hours. After their surfaces were properly cleaned, twelve spots were selected on the surface of neck-portion. These spots were tested by means of the testing machine described above in the direction of growth. The vertical and horizontal magnification were respectively 2500 and 100 and c) Roughness readings Roughness readings have been taken in terms of 'Hmax' and 'Hrme' which are briefly explained as follows (Fig. 3, 4.). Although 'Hmax' is used in the general mechanical engineering for the reading of roughness, it is not sufficient in complicated cases like the present test in which the surface of vital bodies is to be studied. Therefore limax

5 Fig. 3 has been supplemented in the present testing by what is known 'Hrme' for mulated by Fujii in Hmax is obtained according to the equation as indicated by Fig. 3 (A). As shown by the chart, parallel lines were drawn touching more than three of the highest peak and each point. The intervals between them were measured by means of slide calipers. When the readings were divided by the magnification, the roughness (Hmax) was obtained. Hrme' is obtained based on the 'Hmax' readings. This ' method is derived from the thinking that the Hmax readings are considered to be simple periodical curves and the harmonic function is obtained from them. As a result, the roughness curves thus obtained were divided into twelve parts following the suit of C. Runge (1903) who had shown the twelve equal distances on the coordinate axis. Fig. 4 The total length of these curves has been designated the X axis which is divided into the twelve equal portions, and the lines crossing them have been designated as the Y axis. As is shown on Fig. 4 (B), the mean curve C-L is drawn in the center of the peaks of various curves. The node where C-L is crossed by the Y axis is designated a and the node where the roughness curve crosses the Y axis is designated a'. The value in terms of a distance between a and a' is obtained as M and by this means, twelve M values are obtained on the total length of X. This M value which is obtained from the following equation is 'roughness' Hrme.

6 241 In the present experiment, the horizontal magnification has been set at the unit of mm. As the revolution speed of the forwarding bed pulley is 1 mm per minute and 2 mm on the object was magnified to be 100, the length of X curve 16.6 was 200 mm with the distance between the coordinate axes being =0.166 mm. /100 In order to arrive at the readings as accurately as possible, twelve spots were examined on individual teeth and the average was struck on an experiment group of twelve mice. Likewise, the axial magnification has been set at the unit of mm and it was magnified as large as 2500 to the same degree of magnification as the electron microscope. III SUMMARIES AND CONSIDERATIONS The following considerations have been arrived at as results of the series of experiments described above. A. General symptoms A few days following the test of V. C avitaminosis, the general symptom of slight grade. began to appear and in two weeks the symptom was aggravated to the point of diarrhea. After three or four weeks, the majority of test animals were observed to pass into death. When V. C was supplied to these animals by means of subcutaneous injection or oral administration of fresh lemon juice, the general sy.mptom disappeared in two or three weeks depending upon the degree of V. C deficiency. B. Changes in Weight (Fig. 5, 6) Controls animals of g weight showed an average increase of 2.4 g on the daily basis, while the V. C deficient animals showed an average decrease of 2.8 g daily. After three or four weeks, the former showed the total increase of g and the latter the total decrease of g. Fig. 5

7 242 Fig. 6 When they were supplied with V. C, those supplied by means of injection showed the daily recovery of 4.2 g and those of fresh lemon juice showed the daily recovery of 3.8 g. When a comparison was made between the deficient groups of two and three weeks standing, the daily recovery of, the latter was observed to be slightly slower than that of the former. The reason why the recovery by means of oral administration is generally slower than that of the injection may be due to the intestinal absorption. C. Local symptom After two weeks following the V. C deficient test, scorbutic change began to appear in the paradental tissues and at the same time teeth showed the decrease in their color and lucidity changing into the state of cloudiness or milkwhite and sometimes yellowish brown color. After three weeks, teeth were lax in their positions almost to the point of dislocation. Teeth surface began to be quite rough after two or three weeks. When V. C was restored either by injection or oral administration of lemon juice, the scorbutic change in the paradental tissues was recovered in two weeks and after three weeks the lucidity and normal color of teeth were completely recovered with the usual surface. In the case of injection, the recovery was quicker by about five days than the case of oral administration. D. Growth rate of lower incisors (Fig. 7) Compared with the control animals, the lower incisors of experimental animals showed the daily decrease of 0.18 mm in their growth rate and after three or four weeks their growth was completely stopped. In other words, the growth rate decreases in proportion to the advancement of V. C deficiency. When V. C was supplied, the growth was restored by 0.24 mm daily. After

8 243 Fig. 7 four weeks of V. C supply, the growth rate became the normal one as shown by the control animals. E. Electron microscope findings (Fig. 8, 9) While the surface of normal teeth taken from the control animals appeared to be compact and smooth under the electron microscope, obvious roughness of plicate, alveolar, squamous and fissured aspects and imperfect calcification were observed on the surface of V. C deficient teeth under electron microscopy. The degree of this surface roughness takes place in parallel with the advancement of V. C deficiency. With the supply of V. C, the surface was completely restored to the normal state after three or four weeks. F. Roughness readings (Fig. 10) of normal enamel surface of teeth of the control animals, Hmax readings of V. C deficient animals after two or three weeks were the degree of roughnees depending upon the length of its deficienc y When V. C was supplied, the enamel surface gradually began to assume its normal lubricity and density and color, attaining to the normal condition of

9 244 Fig. 10 in terms of proportional numerical value, the corresponding value of V. C defi cient teeth after two or three weeks is somewhere from 7.5 to 9.5. After V. C restoration for five weeks, the recovery takes place at the value of 1.7. IV CONCLUSIONS The following conclusions have been obtained in connection with a series of tests in which the deficiency of V. C is artificially introduced and restored in the teeth of experimental guinea pigs. 1) Compared with the control teeth, the experimental teeth have shown the advanced state of surface roughness and decalcifying disturbance under the electron microscopy. The degree of this surface roughness advances in proportion to the length of time in which V. C is deficient. 2) When the sufficient quantity of V. C is supplied to these deficient teeth, they are brought back to the normal conditions. 3) When the roughness readings of enamel surface of teeth of the control group, V. C deficient and V. C supplied group are measured in terms of proportional numerical value, control teeth show the value of 1.0, and V. C deficient teeth show the value of The same value of the V. C supplied teeth is ) In the restoration of V. C, the injectional administration induces a qui cker restoration than that of oral administration of fresh lemon juice. 5) The macroscopic examinations of teeth of C-Avitaminosis-group in regard to their local symptoms, changes in weight, growth rate, lucidity and color are found to show findings which are more or less in parallel to those of the electron microscopy.

10 245 Fig. 8 Electron-microscopic from I III V VII Normal C-Avitaminosis teeth Findings of EnUmel Surface arising of 1 w. 2 w. ( 2500) II of 1 w. IV of of 2 w. VI of 3 w, of 3 w. VIII of 3 w.

11 246 Fig, 9 Electron-microscopic by I III V VII V. C V, C V. C V. C 1 w. 2 w, 3 w. 4 w. Findings of V. of C. Enamel Surface observed ( 2500) II V. C 1 w. IV V. C 2 w. VI V. C 4 w, VIII V. C 5 w.