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Cell division and periodontal ligament formation in the mouse
Arch oral Biol. Vol. 17, pp. 1781-1784, 1972. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATIONS CELL DIVISION AND PERIODONTAL LIGAMENT FORMATION IN THE MOUSE A. R. TEN CATE Faculty of Dentistry, 124 Edward Street, Toronto 101, Canada Summary-Twelve-day-old mice were injected with 5 &i/g body weight of tritiated thymidine and killed 3,24 and 72 hr after injection. Autoradiographs were prepared of serial bucco-lingual sections through the developing ligament of the first mandibular molar. Analysis of the autoradiographs showed that active cell division occurred in the loose connective tissue of the dental follicle immediately in advance of its organization into periodontal ligament. Electron microscopic examination of this area revealed that cell division was confined to perivascular cells.
As THE periodontal ligament forms, there is an abrupt transition from a loose connective tissue containing young fibroblasts and minimal extracellular collagen to a highly organized connective tissue consisting of actively functioning fibroblasts and dense extracellular bundles of collagen fibres (FREEMAN’ and TEN CATE, 1971). The actively synthesizing fibroblasts were shown to be derived from the maturation of the young fibroblasts of the follicular tissue and from perivascular cells associated with the prominent blood vessels in the transitional zone. An unknown factor is whether or not this change is accomplished by the maturation and hypertrophy of pre-existing cells only, or whether there is also an increase in the cell population due to cell division. To resolve this question, lZday-old mice were utilized as follows. Six animals were given 5 &i/g body weight of tritiated thymidine intraperitoneally. Two of these animals were killed 3 hr after injection, 2 after 24 hr and the remaining 2 after 72 hr. After killing, the lower jaws were removed, fixed in 10 per cent formalin and demineralized in formic acid. Following routine histological procedures, 6-pm bucco-lingual serial sections were prepared through the mesial root of the first mandibular molar. The slides were dipped in Kodak NTB 2 emulsion, exposed for 44 days and then stained with haematoxylin and eosin. Analysis of the autoradiographs was undertaken as follows. Sections were examined until the point was reached where the plane of section included the apical region of the developing molar. From this point on, drawings were made of each section and the position of every labelled nucleus found in the developing ligament recorded. Approximately 40 sections of each specimen were examined. A drawing was then made of a representative mid-series section on which the position of every labelled cell previously recorded was indicated. In this way, a representation was gained of the approximate position of labelled cells within the developing ligament. Figure 1 is a master diagram showing the position of labelled cells within the developing periodontal ligament in a specimen killed 3 hr after injection of tritiated thymidine. 1781 A.O.B. 17/12-K
1782
A. R. TEN CATE
FIG. 1. Master diagram showing the distribution of labelled cells in the 12-day mouse molar developing periodontal ligament 3 hr after Iabelling. The majority of the labelled cells (74 per cent) occur in the unorganized follicle immediately in advance of the forming front (indicated by the oblique lines).
The position of the transitional zone is marked by the oblique lines. The majority of labelled cells occupy a position just apical to the transitional zone. Twenty-four hours after injection the majority of labelled cells were found within the transitional zone and 72 hr after injection of tritiated thymidine, the labelled cells mostly occupied a position within the periodontal ligament occlusal to the transitional zone
2816 T/ I2 day per 72 hr
FIG. 2. Master diagram showing the distribution of labelled cells in the 15-day mouse molar developing periodontal ligament. The label was given 72 hr previously. The majority of labelled cells (78 per cent) are now located within the formed ligament. The forming front is indicated by the oblique lines.
CELL
DIVISION
AND
PERIODONTAL
LIGAMENT
FORMATION
IN THE MOUSE
1783
(Fig. 2). These findings were consistent in both specimens prepared for each time period. From these findings, it can be concluded that the majority of labelled cells occur in the unorganized follicular tissue immediately preceding organization of the ligament and that, with continued root formation, these labelled cells become incorporated into the forming ligament. With the resolution of the light microscope, it is not possible to determine which cells in the unorganized follicular tissue are dividing. To establish this, recourse was made to the material utilized for the ultrastructural study of periodontal ligament development (FREEMANand TEN CATE, 1971). Additional ultrathin sections of 1Zday mouse molar ligament in the transitional zone were cut from the previously prepared blocks, stained with uranyl acetate and lead citrate and examined with a Philips 200 electron microscope. Whilst sampling is always a problem with electron microscopy, in the examination of over 100 grids, each containing on average 3 sections, mitotic figures were found only in cells located in the unorganized follicle preceding the transitional zone. Furthermore, all such cells were always situated perivascularly (Fig. 3). The two main theories concerning the origin of fibroblasts have been well reviewed by VAN WINKLE, JR. (1967) and summarized by MELCHERand EASTOE(1969). It is thought that they have either a haematogenous origin or that they arise locally from undifferentiated mesenchymal cells in loose connective tissue, particularly those cells associated with blood vessels. The ability to demonstrate cell division only in perivascular cells in this study would appear to support the second contention, although the question is then raised as to the origin of the perivascular cells as they may have arisen originally from a haematogenous source. However, previous studies from this laboratory (TEN CATE, MILLS and SOLOMON,1971; TEN CATEand MILLS, 1972) have shown that the tissues of the supporting apparatus, that is cement, periodontal ligament fibroblasts and alveolar bone, are all derived from a thin layer of ectomesenchyma1 cells which, at the cap stage of development, surrounds the dental organ and which is continuous with dental papilla. Thus the perivascular cells must also be derived from this source and therefore a haematogenous source of periodontal ligament fibroblasts seems unlikely. It is suggested, therefore, that the development of the periodontal ligament involves active recruitment of new fibroblasts by division of perivascular cells rather than, or in addition to, the maturation of pre-existing cells within the dental follicle. Acknowledgements-The excellent technical assistance of G. PUDY and D. L. WAGNER is acknowledged. Financial support for this study came from Medical Research Council of Canada, Grant No. MT 3693. R&urn&--Des souris de vingt jours sent injectees a l’aide de 5 &i/g de poids de thymidine tritiee et sacrifices 3, 24 et 72 heures apres l’injection. Des autoradiographies sont preparks a partir de coupes seriees vestibulo-linguales du ligament de la premiere molaire inferieure, en voie de developpement. L’examen des autoradiographies montre qu’une division cellulaire active s’observe dam le tissu conjonctif l&he du follicule dentaire,
CELL
DIVISION
AND
PERIODONTAL
LIGAMENT
FORMATION
IN THE MOUSE
FIG. 3. Electron micrograph of a perivascular cell situated in the loose follicular tissue advance of the forming ligament. This cell is undergoing mitotic division. I - lumen oi vessel, e -7 endothelial cell. 9720
PLArE
1
A.O.B. f.p. 1784
1784
A. R. TEN CATE peu de temps avant sa transformation en ligament alveolo-dentaire. L’examen au microscope tlectronique de cette region demontre que les divisions cellulaires sont limitees aux cellules ptrivasculaires. Zusammenfassung-Zwolf Tage alten Mlusen wurde 5 &i/g Kijrpergewicht TritiumThvmidin iniiziert: die Tiere wurden 3. 24 und 72 Stunden nach Iniektion get&et. Von bu&olinguaien Serienschnitten durch die sich entwickelnden Aufh&gefase& des ersten unteren Molaren wurden Autoradiographien angefertigt. Die Auswertung ergab, dal3 eine aktive Zellteilung im lockeren Bindegewebe des Zahnslckchens unmittelbar vor seiner Organisation zum parodontalen Halteapparat auftrat. Die elektronenmikroskopische Untersuchung dieses Bezirkes machte klar, daR die Zellteilung auf die perivaskularen Zellen beschrankt war. REFERENCES
FREEMAN,E. and TEN CATE, A. R. 1971. Development of the periodontium: An electron microscopic study. J. Period. 42, 387-395. MELCHER,A. H. and EASTOE,J. E. 1969. The Biology of the Periodontium (edited by MELCHER,A. H., and BOWEN,W. H.), Chapter 6, p. 291. Academic Press, New York. TEN CATE, A. R., MILLS, C. and SOUIMON,G. 1971. The development of the periodontium. A transplantation and autoradiographic study. Anut. Rec. 170, 365-380. TEN CATE, A. R. and MILLS, C. 1972. The development of the periodontium: The origin of alveolar bone. Anat. Rec. 173,69-78. VAN WINKLE, W. JR., 1967. The fibroblast in wound healing. Surg. Gyn. Obst. 124, 369-386.
SHORT COMMUNICATIONS CELL DIVISION AND PERIODONTAL LIGAMENT FORMATION IN THE MOUSE A. R. TEN CATE Faculty of Dentistry, 124 Edward Street, Toronto 101, Canada Summary-Twelve-day-old mice were injected with 5 &i/g body weight of tritiated thymidine and killed 3,24 and 72 hr after injection. Autoradiographs were prepared of serial bucco-lingual sections through the developing ligament of the first mandibular molar. Analysis of the autoradiographs showed that active cell division occurred in the loose connective tissue of the dental follicle immediately in advance of its organization into periodontal ligament. Electron microscopic examination of this area revealed that cell division was confined to perivascular cells.
As THE periodontal ligament forms, there is an abrupt transition from a loose connective tissue containing young fibroblasts and minimal extracellular collagen to a highly organized connective tissue consisting of actively functioning fibroblasts and dense extracellular bundles of collagen fibres (FREEMAN’ and TEN CATE, 1971). The actively synthesizing fibroblasts were shown to be derived from the maturation of the young fibroblasts of the follicular tissue and from perivascular cells associated with the prominent blood vessels in the transitional zone. An unknown factor is whether or not this change is accomplished by the maturation and hypertrophy of pre-existing cells only, or whether there is also an increase in the cell population due to cell division. To resolve this question, lZday-old mice were utilized as follows. Six animals were given 5 &i/g body weight of tritiated thymidine intraperitoneally. Two of these animals were killed 3 hr after injection, 2 after 24 hr and the remaining 2 after 72 hr. After killing, the lower jaws were removed, fixed in 10 per cent formalin and demineralized in formic acid. Following routine histological procedures, 6-pm bucco-lingual serial sections were prepared through the mesial root of the first mandibular molar. The slides were dipped in Kodak NTB 2 emulsion, exposed for 44 days and then stained with haematoxylin and eosin. Analysis of the autoradiographs was undertaken as follows. Sections were examined until the point was reached where the plane of section included the apical region of the developing molar. From this point on, drawings were made of each section and the position of every labelled nucleus found in the developing ligament recorded. Approximately 40 sections of each specimen were examined. A drawing was then made of a representative mid-series section on which the position of every labelled cell previously recorded was indicated. In this way, a representation was gained of the approximate position of labelled cells within the developing ligament. Figure 1 is a master diagram showing the position of labelled cells within the developing periodontal ligament in a specimen killed 3 hr after injection of tritiated thymidine. 1781 A.O.B. 17/12-K
1782
A. R. TEN CATE
FIG. 1. Master diagram showing the distribution of labelled cells in the 12-day mouse molar developing periodontal ligament 3 hr after Iabelling. The majority of the labelled cells (74 per cent) occur in the unorganized follicle immediately in advance of the forming front (indicated by the oblique lines).
The position of the transitional zone is marked by the oblique lines. The majority of labelled cells occupy a position just apical to the transitional zone. Twenty-four hours after injection the majority of labelled cells were found within the transitional zone and 72 hr after injection of tritiated thymidine, the labelled cells mostly occupied a position within the periodontal ligament occlusal to the transitional zone
2816 T/ I2 day per 72 hr
FIG. 2. Master diagram showing the distribution of labelled cells in the 15-day mouse molar developing periodontal ligament. The label was given 72 hr previously. The majority of labelled cells (78 per cent) are now located within the formed ligament. The forming front is indicated by the oblique lines.
CELL
DIVISION
AND
PERIODONTAL
LIGAMENT
FORMATION
IN THE MOUSE
1783
(Fig. 2). These findings were consistent in both specimens prepared for each time period. From these findings, it can be concluded that the majority of labelled cells occur in the unorganized follicular tissue immediately preceding organization of the ligament and that, with continued root formation, these labelled cells become incorporated into the forming ligament. With the resolution of the light microscope, it is not possible to determine which cells in the unorganized follicular tissue are dividing. To establish this, recourse was made to the material utilized for the ultrastructural study of periodontal ligament development (FREEMANand TEN CATE, 1971). Additional ultrathin sections of 1Zday mouse molar ligament in the transitional zone were cut from the previously prepared blocks, stained with uranyl acetate and lead citrate and examined with a Philips 200 electron microscope. Whilst sampling is always a problem with electron microscopy, in the examination of over 100 grids, each containing on average 3 sections, mitotic figures were found only in cells located in the unorganized follicle preceding the transitional zone. Furthermore, all such cells were always situated perivascularly (Fig. 3). The two main theories concerning the origin of fibroblasts have been well reviewed by VAN WINKLE, JR. (1967) and summarized by MELCHERand EASTOE(1969). It is thought that they have either a haematogenous origin or that they arise locally from undifferentiated mesenchymal cells in loose connective tissue, particularly those cells associated with blood vessels. The ability to demonstrate cell division only in perivascular cells in this study would appear to support the second contention, although the question is then raised as to the origin of the perivascular cells as they may have arisen originally from a haematogenous source. However, previous studies from this laboratory (TEN CATE, MILLS and SOLOMON,1971; TEN CATEand MILLS, 1972) have shown that the tissues of the supporting apparatus, that is cement, periodontal ligament fibroblasts and alveolar bone, are all derived from a thin layer of ectomesenchyma1 cells which, at the cap stage of development, surrounds the dental organ and which is continuous with dental papilla. Thus the perivascular cells must also be derived from this source and therefore a haematogenous source of periodontal ligament fibroblasts seems unlikely. It is suggested, therefore, that the development of the periodontal ligament involves active recruitment of new fibroblasts by division of perivascular cells rather than, or in addition to, the maturation of pre-existing cells within the dental follicle. Acknowledgements-The excellent technical assistance of G. PUDY and D. L. WAGNER is acknowledged. Financial support for this study came from Medical Research Council of Canada, Grant No. MT 3693. R&urn&--Des souris de vingt jours sent injectees a l’aide de 5 &i/g de poids de thymidine tritiee et sacrifices 3, 24 et 72 heures apres l’injection. Des autoradiographies sont preparks a partir de coupes seriees vestibulo-linguales du ligament de la premiere molaire inferieure, en voie de developpement. L’examen des autoradiographies montre qu’une division cellulaire active s’observe dam le tissu conjonctif l&he du follicule dentaire,
CELL
DIVISION
AND
PERIODONTAL
LIGAMENT
FORMATION
IN THE MOUSE
FIG. 3. Electron micrograph of a perivascular cell situated in the loose follicular tissue advance of the forming ligament. This cell is undergoing mitotic division. I - lumen oi vessel, e -7 endothelial cell. 9720
PLArE
1
A.O.B. f.p. 1784
1784
A. R. TEN CATE peu de temps avant sa transformation en ligament alveolo-dentaire. L’examen au microscope tlectronique de cette region demontre que les divisions cellulaires sont limitees aux cellules ptrivasculaires. Zusammenfassung-Zwolf Tage alten Mlusen wurde 5 &i/g Kijrpergewicht TritiumThvmidin iniiziert: die Tiere wurden 3. 24 und 72 Stunden nach Iniektion get&et. Von bu&olinguaien Serienschnitten durch die sich entwickelnden Aufh&gefase& des ersten unteren Molaren wurden Autoradiographien angefertigt. Die Auswertung ergab, dal3 eine aktive Zellteilung im lockeren Bindegewebe des Zahnslckchens unmittelbar vor seiner Organisation zum parodontalen Halteapparat auftrat. Die elektronenmikroskopische Untersuchung dieses Bezirkes machte klar, daR die Zellteilung auf die perivaskularen Zellen beschrankt war. REFERENCES
FREEMAN,E. and TEN CATE, A. R. 1971. Development of the periodontium: An electron microscopic study. J. Period. 42, 387-395. MELCHER,A. H. and EASTOE,J. E. 1969. The Biology of the Periodontium (edited by MELCHER,A. H., and BOWEN,W. H.), Chapter 6, p. 291. Academic Press, New York. TEN CATE, A. R., MILLS, C. and SOUIMON,G. 1971. The development of the periodontium. A transplantation and autoradiographic study. Anut. Rec. 170, 365-380. TEN CATE, A. R. and MILLS, C. 1972. The development of the periodontium: The origin of alveolar bone. Anat. Rec. 173,69-78. VAN WINKLE, W. JR., 1967. The fibroblast in wound healing. Surg. Gyn. Obst. 124, 369-386.