Arch\ oral Btol. Vol. 24. pp 939 to 943 Pergamon Press Ltd 1980. Prmted m Great Britain
ON THE DAILY INCREMENTAL HUMAN DENTINE
LINES IN
K. KAWASAKI, S. TANAKA and T. ISHIKAWA Department of Anatomy, School of Dental Medicine, Tsurumi University, Z-l-3 Tsurumi, Tsurumi-Ku, Yokohama, Japan Summary-Assuming that the cross-striations on enamel rods represent daily increments, the various incremental lines in dentine between selected pairs of tetracycline markers were identified and correlated with Retzius lines in the enamel, in order to obtain comparative information on the rate of dentine formation. The incremental lines described by Andresen (1898) and von Ebner (1906) are the same and are separated by approx. 20pm. however, a regular series of lines approx. 4 pm apart were also found and considered to be due to a 24-h rhythm in organic matrix formation. A separate 12-h rhythm influencing mineralization was deduced by comparing the number of calcospherite striae with the 4 pm rhythmic change in fibre orientation of the matrix.
enamel may vary, there is reason to believe that the incremental process is less complex than in dentine. According to Boyde (1963), each cross-striation in an enamel rod represents one daily increment of growth. If it is assumed that this is so for a longitudinal section through the mid-plane of a tooth, the cross-striations on the enamel rods could provide an internal control of daily incremental markers over a limited period. By selecting pairs of the tetracycline-marked incremental lines in dentine and following them down to the enamel-dentine junction, the zone of dentine between the origin of the lines would correspond to the zone of enamel between corresponding striae of Retzius in the enamel. Thus, by measuring the interval between the two fluorescent tetracycline lines along the direction of the dentinal tubules in the dentine, and counting the number of cross-striations between the two corresponding striae of Retzius, it is possible to estimate the number of days of dentine formation between the two fluorescent lines (Fig. la). Using this method, it seemed possible to obtain new information on the nature and rate of formation of the various incremental lines in dentine, at least in the crowns of teeth, as the widespread use of tetracycline for a variety of clinical purposes has produced a high incidence of tetracycline fluorescent lines in human permanent teeth.
lNTRODUfflON Although much has been written on the incremental nature of dentine formation, details of the increments and of the mechanisms concerned are still not understood. It is frequently difficult to correlate the observations of different workers as the terms “incremental lines of von Ebner” and the “contour lines of Owen” are not descriptively adequate to distinguish between the numerous striae which can be revealed by various techniques. Some of the confusion arises because the incremental striae in dentine are determined by more than one process, namely the rates at which pre-odontoblast cells are induced to secretory activity and, having commenced secretion, the variation in the rate of secretion during the life cycle of the cells and also the rate at which mineral is deposited into the matrix. On the basis of the work of Schour and his coworkers (1936, 1937, 1939, 1960), it has become generally accepted that dentine forms at an average rate of 16 pm a day for small animals and 4 pm for the macaque monkeys and man. As pointed out by Kawasaki, Tanaka and Ishikawa (1977), the rate of dentine formation is complex and varies with the type of tooth, with the position of the odontoblasts within the tooth, and also the age of the odontoblast as well as being dependent on at least the 3 processes already mentioned. A better understanding of the growth rate characteristics of hard tissue formation is of practical importance to zoologists and forensic scientists. For example, incremental lines in dentine are used to assess the age of seals as a routine procedure in the management and study of seal populations. Incremental lines in cementum are similarly used for studies of deer populations (Morris, 1977). The assessment of the age of the skeletal remains of young children is often an essential stage in the establishment of identity. Cameron and Sims (1974) reported inability to establish the age of a girl of eleven years of age accurately when using the published rate of dentine formation. The enamel of the crowns of teeth shows incremental striae and, although the rate of formation of the
MATERIAL AND METHODS Bucco-lingual longitudinal sections of IO extracted human permanent incisor and premolar teeth which had been immersed in 10 per cent formalin solution were prepared by slicing with a thin Carborundum disc and then grinding and polishing to a final thickness of 30-80 pm. The sectioned teeth were examined by visible transmitted, polarised and U.V. light after lightly etching the enamel briefly with 1 per cent chromic acid (Boyde, 1963). Some specimens were demineralized in 5 per cent nitric acid; some of these were block-stained by Bielschowsky (1946) silver impregnation technique, and thick 12pm frozen sections cut and mounted in Canada Balsam. Frozen sections were cut from the remainder of the specimens and 939
940
K. Kawasaki, S. Tanaka and T. lshikawa
stained with Bielschowsky silver technique. Photomicrographs of selected fields were prepared at a standard magnification ( x 250) and the number of crossstriations on the enamel rods were counted between 2 Retzius lines corresponding with two fluorescent lines in the dentine. Using dividers, the distance was measured between the 2 fluorescent lines along the direction of the dentinal tubules. The daily rate of dentine formation was calculated by dividing the distance in microns between the fluorescent lines by the number of cross-striations. RESULTS
Assuming that each cross-striation on the enamel rods represents one daily increment and using the method outlined above, it was possible to calculate that the incremental lines described independently by Andresen (1898) and von Ebner (1906) are formed every 5 days and are separated by approx. 20pm in the middle part of the cusp dentine (inset la,b). Furthermore when sections stained with silver were examined carefully, 4 extra lines were detected between the Andresen-von Ebner lines. These extra lines, which were about 4pm apart; were also found when both ground and demineralized sections were examined in polarized light. From the polarized light observations, it can be asserted that the Andresenvon Ebner lines and the finer lines between them are due to periodic variations in the orientation of the fibrous matrix during its formation. Mineralization of the dentine follows the establishment of the fibrous matrix and may occur as an epitactic nucleation on the collagen, in which case the mineralizing front appears straight; in addition, socalled calcospheretic mineralization proceeds from centres of crystal nuclei by the periodic deposition of radially oriented crystals. This form of mineralization imparts a globular appearance to the mineralizing front. Bulk staining of dentine with silver before sectioning is capable of revealing very clearly the incremental nature of these spherical-like mineralizations (“Liesegang rings” Mummery, 1919) (Fig. 2). Examination of specimens prepared in this way reveal that there are IO&I2 concentric light and dark laminations (1.7.-2.0 pm apart) which correspond to the distance between two Andresen-von Ebner lines. Thus it would appear that the organic matrix is deposited at the rate of 4 pm every 24 h while the mineral is laid down in the calcospherite at the rate of approx. 2.0 Llrn every I2 h. DISCUSSION
preted the lines of Andresen as being the same as the lines of von Ebner. However. they cited Schour’s 68 pm as the daily rate of apposition of dentine matrix in man and rhesus monkeys and they did not mention that Andresen-von Ebner lines are spaced about 20 pm apart. Our observations using the cross-striation on the enamel rods to estimate the age in days between pairs of tetracycline lines in the dentine are in accord with an average rate of 4 pm per day for the organic phase of dentine formation. However, there are at least 2 other processes concerned with dentine formation; namely, the rate at which new odontoblasts enter their secretory stage, and the rate of mineralization. New odontoblasts in the part of the crown shown in Fig. la must have been added at about 3 times the rate of dentine matrix formation. Figure 2 illustrates the concentric incremental striations which correspond to the periodic mineralization within the calcospherites. These striations are 1.7.-2.0 pm apart and there are I& 12 striations between two adjacent Andresen lines. As the distance between the Andresen lines represents a 5-day period. it may be concluded that the mineralization process follows a 12 hourly rhythm. These results therefore do not appear to correspond with the findings of Miani and Miani (1971) who, using multiple injections of tetracycline, found evidence of a 24 h mineralization rhythm in dogs. However, it is not possible to make a direct comparison between their work and ours; they point out that the circadian rhythm they demonstrated is independent of the quantity of dentine deposited. From our own observations and calculations we conclude that matrix formation has a daily rhythm affecting the orientation of the collagen fibres with a larger 5-day rhythmic change in fibre orientation giving rise to the Andresen-von Ebner lines. The mineralization rhythm on the other hand is I2 hourly. Some confusion still exists because Schour and Hoffman (I 939) refer to an average rate of 16 pm per day for dentine formation in small mammals. They did not refer to the calcospherite striae; thus it is important to investigate the rate of dentine formation in small mammals with the inclusion of preparations capable of demonstrating the so-called “Liesegang ring” phenomenon. AcknowledyrmcwWe express thanks to Professor R. W. Fearnhead of the London Hospital Medical College for his Invaluable help in the preparation of this paper.
REFERENCES
We believe that Andresen (1898) and von Ebner Andresen V. 1898. Die Querstreifung des Dentins. 12. (1906) independently described the same incremental Mschr. Zuhnheilk. 16, 386 389. Bielschowsky M. 1946. In A. Belles Lee’s Microtomlst’s lines in human teeth. These lines are approx. 20pm Vade Mecum 10th Edition. J. & A. Churchill. apart and have 5 sub-divisions each sub-division Boyde A. 1963. Estimation of age at death of young human being separated by about 4pm. which corresponds skeletal remains from incremental lines in the dental reasonably well with the average value of daily increenamel. Third International Meeting in Forensic Immuments of dentine growth reported by &hour and his nology, Medicine, Pathology and Toxicology. co-workers for primate teeth. Thus, as each 4,~~rn Cameron F. M. and Sims B. G. 1974. Forensic Dmti.qfr! increment coincides with a change in the orientation Churchill Livingstone. London. of the fibrous matrix it seems likely that the depositEbner V. von 1906. Uber die Entwicklung der leimgebenion of the organic fibrous matrix is subject to a 24 h den Fibrillen im Zahnbein. Sher. Akad. Wiss. Win;]. 115, rhythm. Orban (1976) and Provenza (1964) interX-349.
On the daily incremental Kawasaki K., Tanaka S. and Ishikawa T. 1977. On the incremental lines in human dentine as revealed by tetracycline labelling. J. Anat. 123, 427436. Miani A. and Miani C. 1971. Circadian advancement rhythm of the calcification front in dog dentine. Mineruu stomat. 20, 169-178. Morris P. 1978. The use of teeth for estimating the age of wild animals. In: Development Function and Evolution of Teeth. (Edited by Butler P. H. and Joysey K. A.) Academic Press. Mummery J. H. 1919. The Microscopic Anatomy of the Teeth. Oxford Medical Publications London. Orban B. J. 1976. Oral Histology and Embryology. Henry Kimpton. London.
Plate
lines in human
dentine
941
Provenza D. V. 1964. Oral Histology. Inheritunce and Development. Pitman Medical, London. Schour I. 1936. The neonatal line in enamel and dentin of the human deciduous teeth and first permanent molar. J. Am. Dent. Ass. 23, 19461955. Schour I. and Poncher H. G. 1937. The rate of apposition of human enamel and dentin as measured by the effects of acute fluorosis. Am. J. Dis. Child. 54, 757-776. Schour I. and Hoffman M. M. 1939. The rate of apposition of enamel and dentin in man and other animals. J. Derlt. Res. 18, 161-175. Schour I. 1960. Noyes’ Oral Histology and Emhryolog~. 8th Ed. Lea & Febiger, Philadelphia.
1 overleaf.
942
K. Kawasaki, S. Tanaka and T. Ishikawa
Plate 1 Fig la. Four tetracycline lines (T) in dentine which originate at the dentineeenamel junction (DEJ) and correspond with striae of Retzius (R) in enamel. Unstained. Ultra-violet fluorescent photomicrograph j’ = approx. 40 pm of dentine formation (10 days) .Y= approx. 1IOpm of new odontoblast differentiation (IO days) u/‘y = 2.75. Insert. A visible light photomicrograph of the same area showing cross-striations between the striae of Retzius (R). Unstained.
(CS) of enamel rods
Fig. I b. An ultra-violet fluorescent photomicrograph in the cusp dentine of the same tooth as Fig. la showing tetracycline lines (T) and the lines of Andresen-von Ebner (AL). Unstained. 4” = 90pm thus V’/V . = 2.25 demonstrating the variations in rate of dentine formation in different parts of the crown dentine (Kawasaki cr al., 1977). From the width of the tetracycline lines, the duration of tetracycline administration can be estimated as 36 days corresponding with standard medical practice. Fig. Ic. A silver-impregnated demineralized section showing Andresen-von Ebner lines (AL) regularly separated by 20 pm. Between these lines, fine striae (arrows) less heavily stained are 4 pm apart. Fig. 2. A section from specimen impregnated with silver before sectioning showing concentric incremental rings or striae (arrowed) 1.7-2.0pm apart. Superimposed on the calcospherite pattern of close lines are the Andresen-von Ebner lines (AL).
On the daily incremental
lines in human dentine
Plate 1.
943
ON THE DAILY INCREMENTAL HUMAN DENTINE
LINES IN
K. KAWASAKI, S. TANAKA and T. ISHIKAWA Department of Anatomy, School of Dental Medicine, Tsurumi University, Z-l-3 Tsurumi, Tsurumi-Ku, Yokohama, Japan Summary-Assuming that the cross-striations on enamel rods represent daily increments, the various incremental lines in dentine between selected pairs of tetracycline markers were identified and correlated with Retzius lines in the enamel, in order to obtain comparative information on the rate of dentine formation. The incremental lines described by Andresen (1898) and von Ebner (1906) are the same and are separated by approx. 20pm. however, a regular series of lines approx. 4 pm apart were also found and considered to be due to a 24-h rhythm in organic matrix formation. A separate 12-h rhythm influencing mineralization was deduced by comparing the number of calcospherite striae with the 4 pm rhythmic change in fibre orientation of the matrix.
enamel may vary, there is reason to believe that the incremental process is less complex than in dentine. According to Boyde (1963), each cross-striation in an enamel rod represents one daily increment of growth. If it is assumed that this is so for a longitudinal section through the mid-plane of a tooth, the cross-striations on the enamel rods could provide an internal control of daily incremental markers over a limited period. By selecting pairs of the tetracycline-marked incremental lines in dentine and following them down to the enamel-dentine junction, the zone of dentine between the origin of the lines would correspond to the zone of enamel between corresponding striae of Retzius in the enamel. Thus, by measuring the interval between the two fluorescent tetracycline lines along the direction of the dentinal tubules in the dentine, and counting the number of cross-striations between the two corresponding striae of Retzius, it is possible to estimate the number of days of dentine formation between the two fluorescent lines (Fig. la). Using this method, it seemed possible to obtain new information on the nature and rate of formation of the various incremental lines in dentine, at least in the crowns of teeth, as the widespread use of tetracycline for a variety of clinical purposes has produced a high incidence of tetracycline fluorescent lines in human permanent teeth.
lNTRODUfflON Although much has been written on the incremental nature of dentine formation, details of the increments and of the mechanisms concerned are still not understood. It is frequently difficult to correlate the observations of different workers as the terms “incremental lines of von Ebner” and the “contour lines of Owen” are not descriptively adequate to distinguish between the numerous striae which can be revealed by various techniques. Some of the confusion arises because the incremental striae in dentine are determined by more than one process, namely the rates at which pre-odontoblast cells are induced to secretory activity and, having commenced secretion, the variation in the rate of secretion during the life cycle of the cells and also the rate at which mineral is deposited into the matrix. On the basis of the work of Schour and his coworkers (1936, 1937, 1939, 1960), it has become generally accepted that dentine forms at an average rate of 16 pm a day for small animals and 4 pm for the macaque monkeys and man. As pointed out by Kawasaki, Tanaka and Ishikawa (1977), the rate of dentine formation is complex and varies with the type of tooth, with the position of the odontoblasts within the tooth, and also the age of the odontoblast as well as being dependent on at least the 3 processes already mentioned. A better understanding of the growth rate characteristics of hard tissue formation is of practical importance to zoologists and forensic scientists. For example, incremental lines in dentine are used to assess the age of seals as a routine procedure in the management and study of seal populations. Incremental lines in cementum are similarly used for studies of deer populations (Morris, 1977). The assessment of the age of the skeletal remains of young children is often an essential stage in the establishment of identity. Cameron and Sims (1974) reported inability to establish the age of a girl of eleven years of age accurately when using the published rate of dentine formation. The enamel of the crowns of teeth shows incremental striae and, although the rate of formation of the
MATERIAL AND METHODS Bucco-lingual longitudinal sections of IO extracted human permanent incisor and premolar teeth which had been immersed in 10 per cent formalin solution were prepared by slicing with a thin Carborundum disc and then grinding and polishing to a final thickness of 30-80 pm. The sectioned teeth were examined by visible transmitted, polarised and U.V. light after lightly etching the enamel briefly with 1 per cent chromic acid (Boyde, 1963). Some specimens were demineralized in 5 per cent nitric acid; some of these were block-stained by Bielschowsky (1946) silver impregnation technique, and thick 12pm frozen sections cut and mounted in Canada Balsam. Frozen sections were cut from the remainder of the specimens and 939
940
K. Kawasaki, S. Tanaka and T. lshikawa
stained with Bielschowsky silver technique. Photomicrographs of selected fields were prepared at a standard magnification ( x 250) and the number of crossstriations on the enamel rods were counted between 2 Retzius lines corresponding with two fluorescent lines in the dentine. Using dividers, the distance was measured between the 2 fluorescent lines along the direction of the dentinal tubules. The daily rate of dentine formation was calculated by dividing the distance in microns between the fluorescent lines by the number of cross-striations. RESULTS
Assuming that each cross-striation on the enamel rods represents one daily increment and using the method outlined above, it was possible to calculate that the incremental lines described independently by Andresen (1898) and von Ebner (1906) are formed every 5 days and are separated by approx. 20pm in the middle part of the cusp dentine (inset la,b). Furthermore when sections stained with silver were examined carefully, 4 extra lines were detected between the Andresen-von Ebner lines. These extra lines, which were about 4pm apart; were also found when both ground and demineralized sections were examined in polarized light. From the polarized light observations, it can be asserted that the Andresenvon Ebner lines and the finer lines between them are due to periodic variations in the orientation of the fibrous matrix during its formation. Mineralization of the dentine follows the establishment of the fibrous matrix and may occur as an epitactic nucleation on the collagen, in which case the mineralizing front appears straight; in addition, socalled calcospheretic mineralization proceeds from centres of crystal nuclei by the periodic deposition of radially oriented crystals. This form of mineralization imparts a globular appearance to the mineralizing front. Bulk staining of dentine with silver before sectioning is capable of revealing very clearly the incremental nature of these spherical-like mineralizations (“Liesegang rings” Mummery, 1919) (Fig. 2). Examination of specimens prepared in this way reveal that there are IO&I2 concentric light and dark laminations (1.7.-2.0 pm apart) which correspond to the distance between two Andresen-von Ebner lines. Thus it would appear that the organic matrix is deposited at the rate of 4 pm every 24 h while the mineral is laid down in the calcospherite at the rate of approx. 2.0 Llrn every I2 h. DISCUSSION
preted the lines of Andresen as being the same as the lines of von Ebner. However. they cited Schour’s 68 pm as the daily rate of apposition of dentine matrix in man and rhesus monkeys and they did not mention that Andresen-von Ebner lines are spaced about 20 pm apart. Our observations using the cross-striation on the enamel rods to estimate the age in days between pairs of tetracycline lines in the dentine are in accord with an average rate of 4 pm per day for the organic phase of dentine formation. However, there are at least 2 other processes concerned with dentine formation; namely, the rate at which new odontoblasts enter their secretory stage, and the rate of mineralization. New odontoblasts in the part of the crown shown in Fig. la must have been added at about 3 times the rate of dentine matrix formation. Figure 2 illustrates the concentric incremental striations which correspond to the periodic mineralization within the calcospherites. These striations are 1.7.-2.0 pm apart and there are I& 12 striations between two adjacent Andresen lines. As the distance between the Andresen lines represents a 5-day period. it may be concluded that the mineralization process follows a 12 hourly rhythm. These results therefore do not appear to correspond with the findings of Miani and Miani (1971) who, using multiple injections of tetracycline, found evidence of a 24 h mineralization rhythm in dogs. However, it is not possible to make a direct comparison between their work and ours; they point out that the circadian rhythm they demonstrated is independent of the quantity of dentine deposited. From our own observations and calculations we conclude that matrix formation has a daily rhythm affecting the orientation of the collagen fibres with a larger 5-day rhythmic change in fibre orientation giving rise to the Andresen-von Ebner lines. The mineralization rhythm on the other hand is I2 hourly. Some confusion still exists because Schour and Hoffman (I 939) refer to an average rate of 16 pm per day for dentine formation in small mammals. They did not refer to the calcospherite striae; thus it is important to investigate the rate of dentine formation in small mammals with the inclusion of preparations capable of demonstrating the so-called “Liesegang ring” phenomenon. AcknowledyrmcwWe express thanks to Professor R. W. Fearnhead of the London Hospital Medical College for his Invaluable help in the preparation of this paper.
REFERENCES
We believe that Andresen (1898) and von Ebner Andresen V. 1898. Die Querstreifung des Dentins. 12. (1906) independently described the same incremental Mschr. Zuhnheilk. 16, 386 389. Bielschowsky M. 1946. In A. Belles Lee’s Microtomlst’s lines in human teeth. These lines are approx. 20pm Vade Mecum 10th Edition. J. & A. Churchill. apart and have 5 sub-divisions each sub-division Boyde A. 1963. Estimation of age at death of young human being separated by about 4pm. which corresponds skeletal remains from incremental lines in the dental reasonably well with the average value of daily increenamel. Third International Meeting in Forensic Immuments of dentine growth reported by &hour and his nology, Medicine, Pathology and Toxicology. co-workers for primate teeth. Thus, as each 4,~~rn Cameron F. M. and Sims B. G. 1974. Forensic Dmti.qfr! increment coincides with a change in the orientation Churchill Livingstone. London. of the fibrous matrix it seems likely that the depositEbner V. von 1906. Uber die Entwicklung der leimgebenion of the organic fibrous matrix is subject to a 24 h den Fibrillen im Zahnbein. Sher. Akad. Wiss. Win;]. 115, rhythm. Orban (1976) and Provenza (1964) interX-349.
On the daily incremental Kawasaki K., Tanaka S. and Ishikawa T. 1977. On the incremental lines in human dentine as revealed by tetracycline labelling. J. Anat. 123, 427436. Miani A. and Miani C. 1971. Circadian advancement rhythm of the calcification front in dog dentine. Mineruu stomat. 20, 169-178. Morris P. 1978. The use of teeth for estimating the age of wild animals. In: Development Function and Evolution of Teeth. (Edited by Butler P. H. and Joysey K. A.) Academic Press. Mummery J. H. 1919. The Microscopic Anatomy of the Teeth. Oxford Medical Publications London. Orban B. J. 1976. Oral Histology and Embryology. Henry Kimpton. London.
Plate
lines in human
dentine
941
Provenza D. V. 1964. Oral Histology. Inheritunce and Development. Pitman Medical, London. Schour I. 1936. The neonatal line in enamel and dentin of the human deciduous teeth and first permanent molar. J. Am. Dent. Ass. 23, 19461955. Schour I. and Poncher H. G. 1937. The rate of apposition of human enamel and dentin as measured by the effects of acute fluorosis. Am. J. Dis. Child. 54, 757-776. Schour I. and Hoffman M. M. 1939. The rate of apposition of enamel and dentin in man and other animals. J. Derlt. Res. 18, 161-175. Schour I. 1960. Noyes’ Oral Histology and Emhryolog~. 8th Ed. Lea & Febiger, Philadelphia.
1 overleaf.
942
K. Kawasaki, S. Tanaka and T. Ishikawa
Plate 1 Fig la. Four tetracycline lines (T) in dentine which originate at the dentineeenamel junction (DEJ) and correspond with striae of Retzius (R) in enamel. Unstained. Ultra-violet fluorescent photomicrograph j’ = approx. 40 pm of dentine formation (10 days) .Y= approx. 1IOpm of new odontoblast differentiation (IO days) u/‘y = 2.75. Insert. A visible light photomicrograph of the same area showing cross-striations between the striae of Retzius (R). Unstained.
(CS) of enamel rods
Fig. I b. An ultra-violet fluorescent photomicrograph in the cusp dentine of the same tooth as Fig. la showing tetracycline lines (T) and the lines of Andresen-von Ebner (AL). Unstained. 4” = 90pm thus V’/V . = 2.25 demonstrating the variations in rate of dentine formation in different parts of the crown dentine (Kawasaki cr al., 1977). From the width of the tetracycline lines, the duration of tetracycline administration can be estimated as 36 days corresponding with standard medical practice. Fig. Ic. A silver-impregnated demineralized section showing Andresen-von Ebner lines (AL) regularly separated by 20 pm. Between these lines, fine striae (arrows) less heavily stained are 4 pm apart. Fig. 2. A section from specimen impregnated with silver before sectioning showing concentric incremental rings or striae (arrowed) 1.7-2.0pm apart. Superimposed on the calcospherite pattern of close lines are the Andresen-von Ebner lines (AL).
On the daily incremental
lines in human dentine
Plate 1.
943
