The Effects of Induced Prenatal Hypothyroidism on Lamb Mandibular Third Primary Molars '92 ROBERT H. BIGGERSTAFF AND JEROME C. ROSE University of Kentucky, College of Dentistry, Department of Orthodontics, Lexington, Kentucky 40536; ' University of Arkansas, Department of Anthropology, Fayetteuille, Arkansas 72701
K E Y WORDS Hypothyroidism Primary Molars
. Intrauterine Thyroidectomies .
Intrauterine thyroidectomies were performed on nine lambs on or about the ninety-sixth postconception day. Seven other control and shamoperated lambs, and the cretin lambs were sacrificed immediately after birth. The mandibles were removed and sectioned at the midline. The right side molars were removed by dissection and caliper measured. The distal cusps of the third primary molars were sectioned, dehydrated, and embedded in Bioplastic. A slow speed diamond saw was used to section the plastic blocks and the embedded teeth. Subsequent grinding and polishing produced high quality 75 pm sections of the lamb molar cusps. No significant differences in tooth size or enamel thickness existed. Microscopic examinations show t h a t parts of the cretin enamel were poorly calcified, an observation that was correlated t o the intrauterine thyroidectomies. The data suggest that hypothyroidism alters ameloblastic activity during the secretory phase of enamel formation.
Dental anthropologists have assumed that features of the dentition are good genetic indicators (Lasker, '50; Kraus, '57; Osborne et al., '58) because few environmental variables are known to interact with the genetic potential of the dentition during growth and maturation. One problem with this simplistic concept is that hormones may represent environmental variables which control cellular activity generally and, therefore, growth. Specifically,the hormone secretions of normal thyroid glands increase the metabolic rate, promote sexual maturation, maintain normal brain and mental development, promote skeletal growth, and maintain normal cutaneous function. These highly interrelated functions suggest that the products of normally functioning thyroid glands are essential for normal body growth and development. The prenatal absence of thyroid secretion, or its severe deficiency, results in the clinical picture of congenital cretinism. The outward manifestations of this condition may include retardation of physical development and delayed eruption of the primary and permanent teeth. It is significant that neural AM. J. PHYS. ANTHROP. (1979)50: 357-362.
growth and mental maturation is retarded also. Intrauterine throidectomies performed on lamb fetuses between the fiftieth and ninetysixth postconception days cause a marked reduction in overall body growth and development a t birth (Hollingsworth et al., '751. Skeletal, neural, and wool development are retarded. Also, mean carcass weight and mean lung weights a r e consistently decreased (Erenberg et al., '74). The retarded developmental patterns of bones, bone tissues, and other tissues of mesenchymal or ectodermal origin are established early in the lives of athyroidic or hypothyroidic individuals. These development trends may continue throughout life (Hollingsworth et al., '75). With the exception of retarded eruption, little is known about t h e effects of hormone deficiencies on the developing dentition. Since tooth morphology results from the interac-
' Presented in part at the annual meeting of the Amencan Associ-
ation of Physical Anthropologist, 12-15 April 1978,Toronto, Ontario, Canada. ?This work was supported by an intramural grant from the University of Kentucky College of Dentistry and by Research Grant CRBS-299from the National Foundation of the March of Dimes.
ROBERT H. BIGGERSTAFF AND JEROME C. ROSE
tions of ectodermal and mesenchymal tissues (Kollar and Baird, '70a,b), it is implied t h a t the absence or deficiency of prenatal hormone will cause prenatal developmental in the newborn lamb mandibular primary molars. Presumably induced hypothyroidism will cause alterations in the by-products of ameloblastic or odontoblastic activity. Therefore, the purpose of this research is to determine t h e effects of induced prenatal hypothyroidism on the enamel and dentin of the newborn lamb mandibular molars. MATERIAL§ AND METHODS
The mandibles of 16 newborn lambs were obtained from ongoing experiments using an intrauterine model designed to study the effects of thyroid function. (The lamb mandibles were made available by Doctor D. R. Hollingsworth, of the University of Kentucky Medical Center.) I n t r a u t e r i n e thyroidectomies were performed on nine lamb fetuses on or about the ninety-sixth postconception day. Seven of these newborn lambs were judged to be cretins by the evaluation of thyroxine blood levels. On the basis of this assay, two other newborn lambs were judged to be partial cretins. Five normal newborn lambs served as controls. In addition, intrauterine sham operations were performed on two pregnant ewes. Each gave birth to a single lamb which served as newborn sham controls. The mandibles were sectioned in the midline. The right halves were used to assess total mandibular molar size. The third primary molars were removed by dissection and measured with Helios dial calipers with readable accuracy to the nearest 0.05 millimeters. The distal cusps of the mandibular third primary molars are the largest; therefore, maximum buccolingual diameters were measured in this area. The teeth were identified by code and shipped to the laboratory of J. C. R. a t the University or Arkansas to be processed for light microscope observations. The distal cuspal components of the mandibular third primary molars were sectioned from the mesial crown components. The distal crown components were dehydrated in successive solutions of ethanol and acetone, then embedded in Bioplastic. Ground and polished sections, approximately 100 p m thick, were prepared by the modified methods of Rose ('77). Each section was etched for 5 seconds with 1N hydrochloric acid, then washed with running water. After air drying each slide was
observed on a ground glass view screen with one photoflood light positioned ten inches below t h e glass and another photo-flood light above the glass a t a 30" angle. A microscale accurate to the nearest 0.1 millimeter was positioned adjacent to the specimen and 35 millimeter black and white negatives were exposed with a Nikon EL camera equipped with bellows and a microlens. Standardized photographic prints included the microscale and specimen enlarged approximately x 7.35. The most cervical point of complete calcification was determined by observing each ground section with a Zeiss microscope at x 250. (Complete calcification occurs when all prisms from the dentino-enamel junction to the enamel surface are etched.) The enamel of this point was compared with the cusp tip enamel and the area was marked on the enlarged photograph. Similarly, the most cervical point where the thickness of enamel shows poor calcification was determined. (Poor calcification occurs when no enamel prisms a r e etched between t h e dentinoenamel junction to the enamel surface.) The enamel of this point was compared with the cusp tip enamel and the area was marked on the enlarged photograph. Measurements from the cusp tip to the most cervical points of complete and poor calcification were recorded. The data were arrayed according to the decoded classifications and analyzed by descriptive statistics. Later the photographs and ground sections were shipped to R.H.B. for independent study. The enamel thickness of the cretin, partial cretin, and control sections was measured on the photographs along a line perpendicular to t h e dentino-enamel junction with needle pointed Helios dial calipers. A one millimeter unit on the photographic image of the microscale was measured and recorded for the reduction of photogrammetric enamel thickness data to normalized metric data. Small sample descriptive statistics and Student's t-test permitted inferences to be drawn. RESULTS
Figure 1 is a photomicrograph of the acid etched enamel of a control lamb mandibular third primary molar. The prisms are arranged in longitudinal rows that tend to follow a straight course. Each prism row is separated by an inter-row sheet intersprismatic substance. The prisms are horseshoe shaped, the open side fitting closely to t h e closed side of its
EFFECTS OF HYPOTHYROIDISM ON LAMB MOLARS
Fig. 1 Control lamb primary molar enamel pattern. The enamel prisms are arranged in longitudinal rows separated by interrow sheets of interprismatic substance.
Fig. 2 Cretin lamb primary molar enamel pattern. Normal enamel (N) prisms and poorly calcified enamel
(P.C.) prisms are observed.
neighbor. This prism packing pattern is typical of the control lamb molar enamel. However, areas of poorly calcified enamel were observed in the most apical regions.
The poorly calcified molar enamel of the cretin lambs is distinguished from normally calcified enamel in figure 2. The interprismatic substance and prism rows are
ROBERT H. BIGGERSTAFF AND JEROME C. ROSE
Fig. 3 Partial cretin lamb molar enamel pattern showing normal (N) and poorly calcified (P.C.) enamel prisms. See text for explanation. TABLE 1
Descriptive statistics of most cervical points along the dentinoenamel junction (in millimeters) of complete and poor calcification of control and cretin newborn lamb molars Complete calcification
x Controls (N = 6) Cretins (N=7)
Partial calcification ~
Complete calcification t a t= 1.69 Partial calcificalion t , , = 2.03
atypical. No longer are the prisms horseshoe shaped. Approximations of normal prism packing are evident and indicate attempts a t achieving the typical pattern. The normal pattern of enamel packing was observed at the cusp tip and cervically along the dentinoenamel junction to a point where poorly calcified enamel was evident. A similar pattern was manifest in the partial cretin molars (fig. 3) with the exception that the poorly calcified enamel appeared better calcified than poorly calcified enamel of cretin lambs. The light microscope observations of poorly calcified and normal enamel were consistent for the cretin and partial cretin lamb molars. Analysis of the measurement data (table 1)
describing the most cervical points of complete calcification shows that the mean length of complete calcification is 2.0 mm for controls and 1.3 mm for cretins. The “t” value 1.69 at 11 d.f. is not significant at the 0.05 level of probability. The normally calcified enamel thickness data (table 2) show no significant differences between cretins and controls. The poorly calcified cretin enamel thickness averaged 0.036 mm whereas the control enamel thickness averaged 0.41 mm. The calculated mean buccolingual dimension was slightly greater for cretin molars (X = 6.84 mm) than the controls CX = 6.51 mm). The differences in mean TABLE 2
Descriptiue statistics of the buccolinguul diameters and thickness measurement (in millimeters) of normal and poorly calcified enamel for control and cretin newborn lamb molars Ruccolingual diameter
x Controls (N = 6 ) Cretins (N=7)
Thickness of Thickness of poorly normal enamel calcified enamel
Buccolingual t , , = 0.868 Poorly calcified enamel t , , = 1.6885
EFFECTS OF HYPOTHYROIDISM ON LAMB MOLARS TABLE 3
Buccolingual diameters and thickness measurements, in millimeters, o f normal and poorly calcified enamel for sham operated and partial cretin newborn lamb mandibular primary molars Bucculingual diameters
Sham operated N=2 Partial cretin N=2
Thickneatl of poorly calcified nurmal enamel enamel
Buccal nurfacr damaged in sectioning and polishing
buccolingual data are not significant and probably occur by chance. Also, the aforementioned calculated buccolingual diameter values are not significantly different from the respective caliper mean dimension data for = 6.64) and cretin lambs control lambs = 6.66). All partial cretin and sham operated lamb measurements are within the range of the control lamb data (table 3). The small sample permits no meaningful statistical comparisons or interpretations.
Induced hypothyroidism may be associated with alterations in enamel formation. The light microscope observations of acid etched ground sections indicate that differences in the quality of normal and cretin lamb enamel exist although no statistical differences were evident between the intergroup measurement data. Enamel and dentin hypoplasias have been significantly correlated to stress and nutritional inadequacy (Giro, '47; Kreshover, '60; Baume and Meyer, '66; Baume and Vulliemoz, '72). The most frequent stress associated form of enamel disturbance is represented by the Neonatal Line (Schour, '361, and accentuated incremental line of Retzius. Its counterpart also occurs in dentin as a Contour Line of Owen. Both are associated with the stress of birth and the process of the neonate adapting to the extra-uterine environment (Via and Churchill, '59). The usefulness of hard tissue defects as research tools may be enhanced by their chronological specificity which permits the assignment of each occurrence to a specific age. Similar enamel defects have been ob-
served in Paleolithic, prehistoric, ancient man (Sognnaes, '55, '56; Rose, '77), and in nonhuman primates (Schuman and Sognnaes, '56; Molnar and Ward, '75). Based on our findings, a key question may be asked. Are we attributing more significance to tooth morphology than is biologically justified? Butler ('56) answered this question succinctly: Since a n adequate understanding of any anatomical structure can be achieved only when its mode of growth is known, it has been difficult for palentologists and others interested in dental morphology to carry their studies beyond the empirical description. The results of this study may add another variable which may have a bearing on our understanding and interpreting tooth morphology. Reductions in cretin lamb skeletal and wool development (Hollingsworth et al., '751, and mean carcass and lung weight (Erenberg et al., '74) may be explained by reductions in the rate and degree of differential growth. Assuming that differential growth is a manifestation of cellular replication, the implication is clear that the cell replication cycle is altered in growing hypothyroidic animals. The aforementioned light microscopic findings show differences in enamel that may be temporally related to induced hypothyroidism. Apparently the stage of partial enamel mineralization is altered most. Other ameloblast functions, e.g., rate of secretion, direction of movement and amount of enamel produced, are relatively unchanged. The progressive nature of good and poor calcification, as observed, shows t h a t the stages of matrix deposition and partial calcification are altered by reduced levels thyroid hormones. Our observations cannot be considered final because of the precise biochemical basis for thyroid hormone functions are incompletely understood. We know t h a t t h e thyroid hormone secretions are composed of two basic components (Ingbar and Weber, '741, thyroxine (TI) and triiodothyronine (T,) and that there are specific hormone target cell receptor sites for each hormone. These receptors may be on the plasma membrane or within the cytoplasm of the target cells, indicating an antigen antibody relationship. These studies may add another dimension to
ROBERT H. BIGGERSTAFF AND JEROME C. ROSE
the analysis of dentitions and t o the understanding of the growth and maturation of the dentition. Unfortunately, studies such as this create severe problems in accepting the findings of more simplistic present day models used to provide specific analytical information related t o dental genetics. LITERATURE CITED Baume, L. J., and J. Meyer 1966 Dental hypoplasia related to malnutrition with specific reference to melanodontia and odontoclauia. J. Den. Res., 45: 426-441. Baume, L. J., and J. P. Vuiliemoz 1972 Variations in the mineral content of Polynesian teeth. Int. Den. J., 22: 193-218. Butler, P. 1956 The ontogeny of the molar pattern. Biol. Rev., 31: 30-70. Erenbera, A., R. Omori, J. H. Menkes, W. Oh and D. A. Fisher- 1974 Growth and Development of the thyroidectoniized ovine fetus. Pediat. Res., 8: 783-789. Giro, C. M. 1947 Enamel hypoplavia in human teeth: An examination of its causes. J. Am. Den. Assn., 34: 310-317. Hollingsworth, D. R., R. P. Belin, J. C. Parker, R. J. Moser and H. McKean 1975 Experimental cretinism in lambs: An intrauterine model with thyroid evaluation in surviving lambs. John Hopkins Med. J., 137: 116-122. Ingar, S. H., and K. A. Weher 1974 The thyroid gland. In: Textbook of Endocrinology. R. H. Williams, ed. W. B. Saunderv Co., Philadelphia, pp. 95-232. Kraus, B. 1957 Genetics of the human dentition. J. Forensic Sciences, 2: 419-427.
Kreshover, S. J . 1960 Metabolic disturbances in tooth formation. Ann. N. Y. Acad. Sci., 85: 161-167. Kollar, E. J., and G. R. Baird 1970a Tissue interactions in embryonic mouse tooth germs. I. Reorganization of the dental epithelium during tooth-germ reconstruction. J. Emhryol. Exp. Morph., 24: 159-171. 1970b Tissue interactions in embryonic mouse tooth germs. 11. The inductive role of the dental papilla. J. Embryol. Exp. Morph., 24: 173-186. Lasker, G. 1950 Genetical analyses of racial traits of the teeth. Sympos. Quant. Biol., 16: 191-203. Molnar, S., and S. C. Ward 1975 Mineral metabolism and microstructural defects in primate teeth. Am. J. Phys. Anthrop., 43: 3-18. Osborne, R., S. Horowitz and F. DeGeorge 1958 Genetic variation in tooth dimensions. A twin study of t h e permanent anterior teeth. Am. J. Human Genet., 10: 350-356. Rose, J. C. 1977 Defective histology of prehistoric teeth from Illinois. Am. J. Phys. Anthrop., 46: 439-446. Schour, I. 1936 The neonatal line in the enamel and dentin of the human deciduous and first permanent molar. J . Am. Den. Assn., 23: 1946-1955. Schuman, E. L., and R. F. Sognnaes 1956 Developmental microscopic defects in the teeth of subhuman primates. Am. J. Phys. Anthrop., 14: 193-214. Sognnaes, R. F. 1955 Post-mortem microscopic defects in the teeth of ancient man. Arch. Path., 59; 559-570. 1956 Histologic evidence of developmental lesions originating from Paleolithic, prehistoric and ancient man. Am. Path., 32: 547-576. Via, W. F., and J. H. Churchill 1959 Relationship of enamel hypoplasia to abnormal events of gestation and birth. J. Am. Den. Assn., 59: 702-707.