Three-dimensional reconstruction of the rat incisor by means of computerized histomorphometry.код для вставкиСкачать
THE ANATOMICAL RECORD 205:455-464 (1983) Three-Dimensional Reconstruction of the Rat Incisor by Means of Computerized Histomorphometry S. STEIGMAN, Y. MICHAELI, M. WEINREB, JR.,AND G. ZAJICEK Department of Orthodontics (S.S.),Anatomy and Embryology WM., M. WJ, and Expeririiental Medicine and Cancer Research (G.Z.), Hebrew Uniuersity-Hadassah Dental and Medical Schools, Jerusalem, Israel ABSTRACT A detailed, quantitative, three-dimensional reconstruction of the adult rat lower incisor is presented. The mandibles of 12 male adult rats were dissected and radiographed. The outline of each incisor was traced and the center of the arc formed by the lower, or labial, border of the tooth was geometrically determined. The sector of the bone-embedded part (mean length 20.70 _+ 3.5 mm) of the tooth was divided into six equal segments by radii drawn through the arc center. The dividing radii were transposed onto the specimens and three consecutive 100-pm transverse ground sections were cut from the incisal side of each segment, parallel to their bordering radii. The distance between each section and posterior wall of the alveolar socket was measured and the information stored in the computer. The outlines of all the dental structures were traced and fed into a computer with the aid of a sonic digitizer. The circumference of the tooth (5.91 0.0021, and the mesiolateral and labiolingual widths (1.36 + 0.001 mm and 2.18 + 0.01 mm, respectively) remained constant at all tooth levels. The decrease in width and cross-sectional area of the pulp and the corresponding increase in dentin were represented by the best fitted second order polynomials. These changes were not uniform, being more pronounced in the proximal part of the tooth. The polynomials also served for calculation of the total volumes of pulp, dentin, and enamel (13.93 mm3, 18.05 mm3, and 4.43 mm3, respectively). The threedimensional measurements included the periodontal ligament (PDL), which was significantly wider on the lateral side than on the mesial (0.16 & 0.006 mm and 0.12 & 0.004 mm, respectively). The volumes of the enamel-bordering PDL and of the cementum-bordering PDL were 7.77 mm3 and 8.67 mm3, respectively. In the latter, the tooth-related compartment constituted 42.7% of the total volume. The data presented can serve as a basis for future quantitative studies of the rat incisor. The constant process of tissue renewal in the rat incisor provides a n excellent model for a study of a variety of fundamental biological problems. Consequently, a profusion of investigations on tooth morphology, with particular stress on the proliferative basal and odontogenic organ, have been and are being carried out. The early works were restricted to two-dimensional studies of tooth morphology, the most detailed one being that of Addison and Appleton (1915). In the 1930s, Schour and Steadman (1935) attempted a three-dimensional generalized description of the rat incisor in connection with the pattern of apposition of dentin and enamel. Later, 0 1983 ALAN R. LISS, INC. more sophisticated approaches employing both longitudinal and cross-sectional planes of reference were applied to the study of the various tooth structures (Pindborg and Weinman, 1959; Chiba, 1965; Robins, 1967; Lavelle, 1968; Moe, 1971; Michaeli and Greulich, 1972; Warshawsky and Smith, 1974; Smith, 1975; Smith and Warshawsky, 1975). In these and other investigations of rat incisor morphology, upper and lower as well as left and right incisors were correlated (Smith and Warshawsky, 1973; Smith, 19751, age Received October 27,1982; accepted January 12, 1983. 456 S. STEIGMAN ET AL. changes explored (Lavelle, 19681, and the incisor compared to the teeth of primates (Warshawsky et al., 1981). The three-dimensional representations of the tooth either lack quantitative data (Smith, 1975; Smith and Warshawsky, 1975, 1976) or concentrate upon the odontogenic organ only (Michaeli and Greulich, 1972). Therefore, in spite of a n abundance of investigations, the basic quantitative data on the various tooth components are still missing. The purpose of the present study is to obtain, by means of a specially designed, computerized image analysis technique, detailed threedimensional quantitative information about the adult rat incisor as a whole and, at the same time, gain data about each of its separate constituent tissues at any given level along the tooth. Fig. 1. Lateral radiograph of a dissected rat mandible. MATERIALS AND METHODS Twelve male adult rats of the Sabra strain (mean weight 230 k 10 g) were used in this experimental work. The mandibles were removed, disssected free of soft tissue, halved through their midline and fixed in BouinHolland fluid. Orthoradial nondistorted lateral radiographs were taken of each mandible (Fig. 1). The fixed mandibular incisors were then divided into six equal segments by a method which eliminates the distortion caused by the tortuous shape of this tooth (Fig. 2). Namely, each radiographed mandibular bone and its tooth were traced on an acetate sheet, and the center of the arc formed by the labial border of the boneembedded part of the incisor was geometrically determined. From this center, two radii were drawn-one to the posterior limit of the alveolar socket, and the second to the ante- Abbreviations b, bone b-pdl, bone-related periodontal ligament d, dentin e, enamel L, lateral Lin, lingual M, mesial n, nerve P! Pulp pdl, periodontal ligament t-pdl, tooth-related periodontal ligament v, blood vessel Fig. 2. Schematic drawing showing the division of the rat incisor into six (a to D equal segments. rior limit of the lingual alveolar bone. The arc of the segment bordered by these two outer radii was measured, and six equal parts were marked off. Radii passing through these points of measurements were drawn, dividing this tooth section into segments of identical size. The left mandibles were mounted on an Isomet low-speed saw (Buhler Ltd.), and three consecutive 100-pmthick undecalcified transverse sections were cut from the incisal side of each of the six segements, parallel to their bordering radii (Fig. 3). The micrometer, which forms a part of the Isomet saw, accurately recorded the thickness of each cut section of the tooth. The length of the remaining sector was measured precisely using a fine caliper connected to a digital voltmeter. These measurements established the exact distance of each ground section from the posterior limit of the alveolar socket. 457 THREE-DIMENSIONAL RAT INCISOR RECONSTRUCTION Fig. 3. Cut cross sections of rat incisor from segments a, b, c, d (see Fig. 2). X 28. 458 S. STEIGMAN ET AL. The sections were viewed on a Reichert Visopane microscope, and the outlines of the tooth components and its surrounding bone were traced. The tracings were fed into a computer with the aid of a sonic digitizer. Each curve was double-traced with a special stylus, and the coordinates of each point were introduced into a PDP-15/20 computer and displayed as digitized tracings on a storage scope for visual inspection. Special programs computed the dimensions of the different anatomical structures. The following cross-sectional parameters were measured (Fig. 4): 1. Total tooth dimensions: circumference with and without the enamel layer; mesiolatera1 width (as expressed by line 1-1' joining the mesial and lateral cementoenamel junctions); labiolingual width (as expressed by line 2-2'). To establish the latter, a point on the lingual tooth outline (2) representing the maximal distance between this outline and the midpoint on the cementoenamel junction plane was determined. The maximal distance between point (2) and the labial outer surface (point 2') was then measured. 2. Pulp: circumference; mesiolateral width along the cementoenamel junction plane; maximal labiolingual width. 3. The width of the dentin measured between its outer and inner (including predentin) boundaries along lines drawn in the L Fig. 4. Tracing showing the location of lines used in the measurements. For detailed explanation, see text. center of the labial, lingual, mesial, and lateral dentinal walls, perpendicular to the outer surface (line 3); width of the enamel (line 4)as measured along the line of maximal tooth width. 4. The periodontal ligament (PDL) was divided by the cementoenamel junction plane into its two anatomically different compartments-i.e., the enamel- and the wmentumbordering ligament, respectively. The width of the former was measured along the median perpendicular to the cementoenamel junction plane. To calculate the mesial and lateral periodontal width, the cementum-bordering PDL was arbitrarily subdivided into mesial, lateral, and lingual sections by a n arc tangent to the outer lingual tooth outline and having as its center the midpoint of the cemento-enamel junction plane. The division of the areas of the mesial and lateral sections by their measured length yielded the periodontal space width. The cementum-bordering PDL was subdivided into its two morphologically different compartments (Fig. 5A)-i.e., the tooth- and the bone-related components (Beertsen, 1973). In order to measure the dimensions of each of these compartments separately, the lower right mandibles were decalcified in 10% EDTA, divided into six segments, using the same technique as for the left incisor, and embedded in paraplast. Sections (6 pm) were cut from the incisal side of each of the six segments, parallel to their bordering radii, and stained with hematoxylin-eosin. The sections were subjected to histomorphometric procedures as described above and, in addition, a demarcation line between the two compartments was drawn (Fig. 5B), enabling their separate evaluation. The most proximal sections of the tooth, which are not completely surrounded by bone, were excluded. The measurements obtained from all ground sections were plotted according to their distance from the posterior wall of the alveolar socket (abscissa), and second-order polynomials were fitted to these points (Michaeli et al., 1981). In these polynomials the value y depicts the tissue parameter dependent upon the distance of the tissue (x) from the tooth origin. The adequacy of the polynomial fit was checked using the x2 test. The polynomials did not deviate significantly from the observed points, thus rendering them valid for the derived computations. The cross-sectional areas of pulp, dentin, and enamel and their total volumes, as well as the volumes of the different parts of PDL, were THREE-DIMENSIONAL RAT INCISOR RECONSTRUCTION 459 Fig. 5. The division of the periodontal ligament into tooth-related and bone-related compartments. A) Histo logical section showing the clearly defined demarcation line (black arrows); B) tracing. Hematoxylin-eosin. X750. computed by specially designed programs. Student’s t-test was used for statistical evaluation of the results. RESULTS In the present investigation the measured and calculated values were obtained from that bone-embedded part of the rat incisor where dentin secretion already fully surrounds the pulp-that is, approximately 4 mm from the posterior alveolar wall. From this point on, the tooth was perfectly cylindrical, presenting identical dimensions of cross-sectional widths and dentin circumference along the entire length (Fig. 6B, C, D). Proximally (9 mm), the total tooth circumference showed a very slight increase, reflecting the process of enamel apposition (Fig. 6A). The tooth dimensions are given in Table 1. Since the dimensions of the pulp reflected the rate of dentinogenesis, these two tooth components will be described together. The cross-sectional dentin area continuously increased, reaching 2 mm2 a t the alveolar border, whereas the corresponding pulp area decreased from 2 mm2 to 0.1 mm2 (Fig. 7A, B). These dimensional changes did not proceed uniformly, being more pronounced proximally (up to 13 mm), with considerable deceleration toward the alveolar crest. The augmentation in the width of the dentinal walls was identical on the mesial, lateral, and labial aspects of the tooth, the last aspect remaining thicker throughout by approximately 50 pm. As already reflected by the areal dimensions, there was a n initial steep increase in dentinal width along the apical half of the tooth, slowing down distally (Fig. 8B). The lingual dentinal wall (Fig. 8A) showed a different picture, as expressed by the polynomial fitted to its measurements. The steep increase in thickness proceeded steadily toward the distal tooth end, culminating in a mean dentinal width of 0.74 mm at the level of the alveolar crest. This was considerably thicker than the mesial dentin (0.51 mm) at the same level (P < 0.001). Consequently, the labiolingual width of the pulp decreased almost uniformly from a mean of 1.9 mm a t the apical end to 0.7 mm at the distal aspect, whereas the decrease in the mesiolateral width (from 1.2 mm to 0.1 460 S. STEIGMAN ET AL. Fig. 6. The overall tooth dimensions. A) Tooth circumference; B) outer dentin circumference; C) cross-sectional mesiolateral width; D) cross-sectional labiolingual width. TABLE 1. The measured and calculated parameters of dental tissues in adult rat incisors Total parameter Tooth Bone-embedded length Circumference Mesiolateral width Labiolingual width Pulp Dentin: circumference Enamel Width (distal 5 mm) Cross-sectional area (distal 5 mm) Periodontal ligament Enamel-bordering Cementum-bordering(tooth-related part) Cementum-bordering (bone-related part) Total mesial width Total lateral width Tooth-related mesial width Tooth-related lateral width Tooth-related lingual width mm) occurred mainly along the proximal 13mm part of the tooth length (Fig. 9). Therefore, the contour of the pulp constantly changed, the ratio of its mesiolateral to labiolingual dimensions diminishing from 0.64 a t the apical end to 0.14 a t the alveolar crest level. The cross-sectional enamel area and Mean size (mm) 20.70 5.91 1.36 2.18 Total volume (mm3) f 0.350 f 0.020 f 0.010 f 0.010 5.53 f 0.030 13.93 18.05 4.43 0.14 f 0.003 0.28 f 0.005 16.44 7.94 3.63 4.87 0.12 f 0.004 0.16 f 0.006 0.052 ? 0.003 0.057 f 0.003 0.105 f 0.006 width continued to increase in the proximal 8 mm of the tooth, after which these parameters remained constant (Fig. 10). The enamel-bordering PDL, which in the rat incisor does not contain tooth anchoring collagenous fibers, exhibited a regular outer border. It was widest a t the proximal tooth THREE-DIMENSIONAL RAT INCISOR RECONSTRUCTION end (0.43 mm), gradually lessening to a constant mean value of 0.18 mm in the distal half of the tooth (Fig. 11). In the cementum-bordering PDL, the outer border and, as a result, the cross-sectional PDL area, varied in the different section. This was particularly evident on the lingual tooth aspect. Therefore, only the total PDL volume and the mean width of the mesial and lateral PDL were estimated (Table 1). 461 The lateral PDL was significantly wider than the mesial one (P < 0.001). The tooth-related PDL constituted 42.7% of the total PDL volume (Table 1).It had fairly regular outlines, with the mesial and lateral sections having a n almost similar width, and the lingual aspect being nearly double their thickness (Table 1; Fig. 12).On all these three sides, the width of the tooth-related PDL, as well as its area (Fig. 13B), increased in the proximal part of the tooth, reaching constant values at about 8-10 mm from the posterior wall of the alveolar socket. The measurements of the bone-related PDL area (Fig. 13A) yielded widely divergent values with a tendency to similarity along the tooth length. DISCUSSION Fig. 7. Graphic presentation and polynomial equation of the cross-sectional area of pulp (A) and dentin (B). y~ = 2.92 - 0 . 2 5 ~ 0 . 0 0 6 ~ YB ~ ; = -0.74 + 0 . 2 3 ~- + 0.005~~. The few three-dimensional reconstructions of the basal part of the rat incisor described in the literature (Michaeli and Greulich, 1972; Smith and Warshawsky, 1975) were executed by the painstaking method of gluing together thin plates of material representing enlarged cross sections of the tooth. The application of computerized image analysis facilitated the development of a new and simpler method for the three-dimensional demonstration of the tooth as a whole, and of each of its tissues separately. The lower rat incisor is difficult to reproduce, as it is both curved anteroposteriorly and twisted mesiolaterally, comparable to "a portion of a flattened spiral" (Addison and -I 0.6 - 0.6 x/'xs X i 0 Fig. 8. Graphic presentation and polynomial equation of the cross-sectional width of the lingual (A) and mesial (B) dentin. y~ = 0.22 + 0 . 0 5 ~- 0.0001~~; YB = -0.18 + 0 . 0 5 ~- 0 . 0 0 0 6 ~ ~ . 462 S. STEIGMAN Fig. 9. Graphic presentation and polynomial equation of the pulp circumference (A) and cross-sectional labiolingual (B) and mesiolateral C) width. y~ = 6.73 - r n d 0.4r 2'0 mrn 15 Fig. 10. Graphic presentation of the cross-sectional area of enamel and polynomial equation for the proximal 8 mm of this tissue. y = -0.09 + 0 . 0 6 ~- 0 . 0 0 1 4 ~ ~ . 0 0.4 .5 t I I I I 5 I 1 I 1 , 1b , I , (b I T 1 1 2 0 mm Fig. 11. Graphic presentation and polynominal equation of the cross-sectional width of the enamel-bordering periodontal ligament. y = 0.53 - 0 . 0 5 ~+ 0 . 0 0 1 7 ~ ~ . ET AL. 0 . 3 1 ~+ 0 . 0 0 3 ~YB ~ ; = 2.40 1.85 - 0.13~+ 0 . 0 0 2 ~ ~ . - 0 . 0 9 ~- 0 . 0 0 0 4 ~yc ~; = Appleton, 1915).We have endeavored to overcome this difficulty by dividing the tooth into relatively small, straight segments and sampling each of these segments perpendicularly to its own axis of length. The degree of accuracy obtained by our method is reflected in the measurements of the total tooth dimensions. As the continuous apposition of dentin progresses inward from the external perimeter of the tooth, the previously established circumference at the proximal tooth end has to retain its shape and size. And, indeed, the outline and width of the teeth measured in our study produced identical values at all levels of sectioning, offering proof that no element of distortion was present. The new method described here enables the measurement and calculations of the parameters of various dental tissues. The amounts of dentinal and pulpal tissues change incessantly from the proximal to the distal tooth extremities, prohibiting calculation of total mean values for these tissues. Thus, each parameter has to be evaluated separately at any given level, using the fitted polynomials. The continuous addition of new dentin and the consequent narrowing of pulp space did not proceed uniformly. To understand this phenomenon, one has to view the distance of any given section of tissue as a function of time. The dentin-secreting cells, maturing near the tooth origin, are passively carried by the tooth eruption toward the distal tooth end, where they die and are eliminated by the process of attrition. As the daily THREE-DIMENSIONAL RAT INCISOR RECONSTRUCTION mm 463 of the values obtained for dentin shows that, starting from approximately the age of 29 days (13 mm distant from the proximal end), the rate of apposition by the older cells is slowed down. This might be explained by either the process of aging of the secreting cells or the environmental changes of the progressive pulp obliteration. An interesting finding was the exceptional behavior of the lingual dentin, which not only failed to show deceleration in the process of its increasing width, but even exhibited a certain accelera0.21 tion. This is a n example of a response to physiological demands upon tissue, as the lingual dentin at the distal tooth end directly bears the impact of the attritional activities of the animal. I " " l " " I " " l " " I ~ 10 15 20 m m The data obtained on enamel width tally with those already published in the literaFig. 12. Graphic presentation and polynomial equature (Addison and Appleton, 1915;Schour and tion for the proximal 8 mm of the cross-sectional width of the lingual (A),mesial (B), and lateral (C) tooth-related Steadman, 1935). However, it has to be periodontal ligament. y~ = 0.13 + 0 . 0 5 ~- 0 . 0 0 3 ~yB ~; pointed out that they represent only the = -0.13 + 0 . 0 5 ~- 0 . 0 0 4 ~ yc ~ ; = 0.14 - 0 . 0 4 ~+ maximal width of the labial surface while 0.003~~. the enamel layer tapers toward the mesial and lateral cementoenamel junction. Warshawsky and Smith (1974) have found that the length of the presecretory and secretory zones of an enamel organ equals 7.2 mm. In keeping with their findings, our results, too, show a secretory activity approximating8 mm in length, as measured from the posterior wall of the alveolar socket; because of its constantly increasing width, the area of enamel in this part of the tooth has to be expressed r n d by its polynomial. 0.51 In the present work, the detailed quantitative investigation of the PDL is an additional 0.4 vy feature in the accumulating knowledge about X the rat incisor. The increment in the dimeno.31 sions of the periodontal space on the lateral x% xx X and lingual sides accounts for the increased tooth mobility in these two directions. It is O-*t interesting to note that, in spite of the difference in the total width of the mesial and lateral PDL, the tooth-related PDL on these O" sides was nearly equal. The increment in the width of the lateral PDL has therefore to be ascribed to the bone-related, blood vesselFig. 13. Graphic presentation of the cross-sectional containing ligament. It has to be rememarea of the bone-related (A) and tooth-related (B) cemenbered that the findings on the PDL in the tum-bordering periodontal ligament and polynomial equations for A and for the proximal 8 mm of B. y~ = present work were obtained on healthy, nor0.24 + 0.02X - o.ooo7X2;yB = -0.13 + 0 . 0 7 ~- 0 . 0 0 4 ~ ~ . mally erupting incisors. Whether these values for bone- and tooth-related PDL are applicable under changed functional derate of eruption of the rat incisor is constant, mands is now under investigation. amounting to 450 pmlday, the division of the The data presented in this study may serve distance of a given section from its basal end as a baseline for future quantitative experiby this value establishes the age of the odonments on the rat incisor. One of the main toblasts in this specific section. Examination merits of the described method is the ability o.2 t 1 L t x--x +-- 464 S. STEIGMAN ET AL. to calculate tissue volumes. As such, it can also be utilized to measure precisely the volumes of fragments of dentin or enamel used for chemical analysis, or to establish tissue volume in any given segment of the tooth. The three-dimensional reconstruction of the dental cell environment, together with the knowledge of daily celI movement (derived from measuring tooth eruption), can provide kinetic information without resorting to the use of isotopes and autoradiography. For example, this method was successfully applied to the study of kinetics of the odontoblast population of the rat incisor (Weinreb et al., 1982) and the rate of dentinogenesis (Michaeli et al., 1982). The computerized histomorphometric method, which in our study was applied to the detailed portrayal of the rat lower incisor (Fig. 14), can be successfully used for the Fig. 14. Example of a computerized three-dimensional reconstruction of lower rat incisor obtained from cross-sectional tracings at various levels along the tooth. quantitative description of any anatomical structure, provided it has a clearly defined shape. If the replicating cells in the structure present a steady state, the method can also be applied to kinetic investigations. LITERATURE CITED Addison, W.H.F., and J.L Appleton Jr. (1915) The structure and growth of the incisor teeth of the albino rat. J. Morphol., 2643-96. Beertsen, W. (1973) Tissue dynamics in the periodontal ligament of the mandibular incisor of the mouse: A preliminary report. Arch. Oral Biol., 18:61-66. Chiba, M. (1965) Cellular proliferation in the tooth germs of the rat incisor. Arch. Oral Biol., 10r707-718. Lavelle, C. (1968)Some age changes in rat incisor teeth. J. Gerontol., 23:393-395. Michaeli, Y., and R.C. Greulich (1972) A three dimensional representation of the odontogenic epithelium of the rat incisor. Anat. Rec., 174r389-398. Michaeli, Y., S.Pitary, and G. Zajicek (1981) On the derivation of cell kinetics from histomorphology, observed in the rat incisor odontoblast. Cell Tissue Kinet., 14:73-84. Michaeli, Y., S.Steigman, M. Weinreb Jr., and G. Zajicek (1982) Histomorphometric study on the impeded and unimpeded rat incisor. In: M. Silberman and H.C. Slavkin, eds. Current Advances in Skeletogenesis. Excerpta Medica, Amsterdam-Oxford-Princeton, pp. 276283. Moe, H. (1971) Morphological changes in the infranuclear portions of the enamel-producing cells during their life cycle. J. Anat., 108:43-62. Pindborg, J.J.,and J.P. Weinman (1959)Morphologic and functional correlation in the enamel organ of the rat incisor during amelogenesis. Acta Anat., 36r367-381. Robins, M.W. (1967) The proliferation of pulp cells in rat incisors. Arch. Oral Biol., 12:487-501. Schour. J.. and S.R. Steadman (1935)The mowth uattern and daily rhythm of the incisor of the r i t . An&. Rec., 6.332.5-333. Smith,C.E., and H. Warshawsky (1973) Radiographic determination of the length of the maxillary and mandibular incisor of the rat. J. Dent. Res., 52:1234-1237. Smith, C.E., and H. Warshawsky (1975) Histological and three dimensional organization of the odontogenic organ in the lower incisor of 100-gram rats. Am. J. Anat., 142403-429. Smith, C.E., and H. Warshawsky (1976) Movement of entire cell populations during renewal of the rat incisor as shown by radioautography after labeling with 3H-thymidine.Am. J. Anat., 145225-260. Warshawsky, H., and C.E. Smith (1974) Morphological classification of rat incisor ameloblasts. Anat Rec., 179423-446. Warshawsky, H., K. Josephson, A. Thylstrup, and 0. Fejerskow (1981) The development of enamel structure in rat incisors, as compared to the teeth of monkey and man. Anat. Rec., 20Or371-399. Weinreb, M. Jr., S. Steigman, Y. Michaeli, and G. Zajicek (1982) Odontoblast turnover in the impeded and unimpeded rat incisor derived from computerized histomorphometry. Arch Oral Biol., (submitted).