Mitotic Potential of the of the Rhesus Monkey

W. 0. ENGLER,* S. P. RAMFJORD, and J. J. HINIKERt School of Dentistry, The University of Michigan, Ann Arbor, Michigan, and Veterans Administration Hospital, Ann Arbor, Michigan

The purpose of the present investigation Although the metabolic activity in sev- was to study the mitotic behavior of the eral highly differentiated cell groups2 6, 7 cells that make up the enamel organs of de- may induce labeling without the cells' going veloping teeth by using tritiated thymidine. into division, it is generally accepted that Recent radioautographic studies with cells which incorporate tritium-labeled thy- tritiated thymidine have enhanced the midine are beginning DNA replication in knowledge of cell-proliferation sites and cell- preparation for mitosis. The labeled cells migration patterns in the developing molar can be recognized by the localization of ex- teeth of the guinea pig,1 rabbit,2 and rat' posed silver grains over their nuclei. In the and in the developing incisor of the mouse.4 present investigation labeled cells will be In order to simulate the conditions of the considered indicative of impending prolifer- developing human as much as pos- ation, as has been the procedure in a number sible, rhesus monkeys were selected for this of recent studies of oral structures.' 3,4 8 12 isotope study. Results Materials and Methods Initiation of the from the The same animals and techniques were ectoderm occurs prior to 4 months of fetal used as those reported in another paper on life. Thus, sections depicting the initiation the development of the epithelial attach- of development were not available. ment.' Tritiated thymidinel (1 Mc/gm of However, from the fetus and the baby mon- body weight; specific activity, 6.7 c/mmole) key numerous sections demonstrated the was administered intravenously to 1 adult various stages of odontogenesis following monkey with a 4-month fetus and intraperi- the formation of the dental lamina. The toneally to a 42-month-old monkey. The present report starts with the enamel organ animals were sacrificed 1 hour after the in- at the stage of a proliferating dental lamina jection. and extends to completed . The Only the jaws of the fetus and baby mon- radioactive index of the fetal oral epithelium key were used for the present study. These was 2.5 per cent (of 12,093 cells counted, were fixed in 10 per cent neutral buffered 303 were labeled) compared with 2.0 per Formalin, decalcified in 20 per cent ethyl- cent for the baby monkey. The fetal oral enediaminetetraacetic acid, imbedded in epithelium was 8-10 cell layers thick and paraffin, and sectioned at 8 Mu in the usual showed labeling in the basal layer (Fig. 1). manner. Radioautographs were prepared by In the 4-month fetus, the dental lamina dipping the slides into NTB-3§ emulsion. appeared as a band of three to four cell They were exposed for 3 weeks in light-proof layers of elongated epithelial cells and had boxes at 4° C., developed, and stained with severed its connection with the overlying hematoxylin. oral epithelium. The lamina was located Received for publication July 9, 1964. within the fetal jaw at the junction of the crypt of * Present address: National Institute of Dental Research, the deciduous tooth and the dermal National Institutes of Health, Bethesda, Md. connective tissue. In sections where an un- t Present address: Veterans Administration, D.C. Washington, differentiated dental lamina was present, t New England Nuclear Corporation, Boston, Mass. labeled cells were occasionally observed at § Eastman Kodak Company, Rochester, N.Y. irregular intervals. 1285 1226 ENCIGIER, 1?A1 UFJOIRIt ANV) /INIRIER .T. dent. XRes. Noeober I--December 1965 As bud formation occurred along the cells of the enamnel organ xN crc labeled at larmina, the cells lining the periphery at the this stage. Labeling was also evident in the site of the bud ,assumed a perpendicular ori- connective tissue of the dental sack sur- entation to the laimina proper and differen- rounding the developing bud. tiated into short cuboidal cells (Fig. 2). The Labeled cells xvere numerous in the epi- cells within the developing bud exhibited thelium of the developing enamel organ and prickle-cell arrangement and appeared more in the surrounding connective tissue as the widely spaced than the peripheral cells (Fig. tooth development proceeded to the cap 3). Malnyvof both the outer and the inner stage (Fig. 4). Three recognizable layers were observed in the epitheliall tissues form- ing the cap. One outer epithelial layer com- posed of tightly packed cuboidal to short columnar cells covered the convexitx of the cap and inclosed more widely spaced polx g- onal cells filling the center of the develop- ing enamel organ. \Within the concavity of the cap, the lining epithelial cells had difer- xe itiC:;EdE: t~iC !!i k; M; ; 'Mm entiated into parallel tall columnar cells. iz ffi^ i'i'9 4 2v(_~~~~~~~ ~ ~ ~~~~~~~~~~~....; ...\; Labeled cells were present in the epithelium along both the outer and inner borders of the cap, and sonme of the central cells were labeled, especially- those adjacent to the inner border epitheliumii. Numerous labeled cells were present in the connective tissue of the uLnder the developing FIeG. 1.--Fetal oral. epitheliun. Labeled cells in enamel organ. basal layer. (flermatox lin stain.) As the differentiation of the epithelial cells of the enaimel organ continued, the four classical layers of the bell stage wxere ob- served. At the junction of the inner and outer enamiiel epithelium, labeling ,appeared

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Fic. 2. Labeled cells in outer and inner cell layers of early-buld-stage enamel organ and in sur- FIG. 3. Labeled cells within center of bud-stage rounding connective tissue. (Hematoxy.7in stain.) enamel organ of monkey fetus. (Hematoxlin stain.) Vol2. -14, \o. 0 V11oTOI7C P1OTEVTIAIL OF iT FE ENA. IEL ORGAN 1287 to oCCu.1r in both lax ers. Elsewhere, labeled As amnelogenesis wxts completed, the amel- cells were absent in the stellate reticulurm, oblasts lost their Tomes's process configura- occasionally seen in the outer enamel epi- tion, and the nuclei of the be- theliuml, and-i firequentl> observed in the cam-ie centrally located and stained less dis- st~rattum internllelium and the inner enamel tinctively with hematoxxvlin. The stratum epithelium (Fig. 5). As the inner enamel intermediumni lost its perlpendicular orienta- epithelium att tlhe future amelodentinld junc- tion to the reduced- laver and tion (at the tip of the cusp) developed into formed a united nondescript layer with the prea-meloblasts, substantial labeling could cells of the outer enamel epiteldiCum. The still be observed. After tientlin--matrix for- combined epithelial layers, that is, the inner matiion xxWas initiatetl, tlhe preameloblasts in reduceda-nmeloblast layer and the outer lay- the immediate area of the newxlx formed er derived from stratum intermeedium and lentin matrix (lifferentiated into amn1elo- outer enamel epitheliumn together formed blasts. Labeling ceased. in the am-eloblast the reduced-ena-mel epit helitum- of the un- laxver as this dlifferentiation occurred. The erupted tooth. Labeling xas absent in the stratum inter-medium was t-hen reduced to a laver of reduced ameloblasts but wxas occa- one-cell lawyer per-pendli(ular to the amelo- sionallx seen in the outer laxer of the re- blasts (Fig. 6). Labeled cells were found in luced-enamel epitheliLlumI fromll Completion I Ithe stratu IinIternied-iIIum thrIoughout amelo- of the amelogenesis until the teeth ,almost genesis. Labeling in the outer Cnalmel CJ)1- reached cont at t Wit h the oral mucosa during thelium w obserIved occasIonallv until eruptio011. completion of aImelogenesis (Fig. 7). The zoneV of tfie stellate reticultum gradu- I iseussion allx (decreasetl in size as the enaiel fori-uma- TFhe ferts, with its rapidly expanding cell tion progressed until it. was no longer visible population, provided an excellent oppor- s a(ldistinct, layeI- (Figs. 8, 9). The cells of tunitx to study cell-behavior patterns dur- the andassociated ing tooth development xvith no interference c'apillaries closely alpproXimaltel the St1'lttumn1 from ext raneous; factors. The passage of tri- interme(lium at this stage (Fiigs. 7-19). L'a- tiatecl thycmidine from the mother to the bele.l cells still wlTre observccl in the sratum fetus at 4 months' gestation wxas efficient intermedim(aLnIl occasionally in the outer since radioauitographs of good quality were enamel epithelial laerl-. obtained from1 the fetus.

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F a,.. 4. t (I)-Sta(geC CenZaIIIC organ of bait monkey Labeled cells in oLuter and inner cell lavers. (Hlema- tox\ in sLain.) 1I. 5.-l-abeled lpreameloldasts, (A;) and (B) in bell-stage enamel organ of baby monkey- StClltC e-CtiCLIIeIIII (C). (Hematoxylin stain.) 1222 ENGTLER, R JItFJORD, AND IIIXVIKER J. dent. Res. Notember-December 1965 11e tooth dlevelopment in the -monkey teeth. The in.Iages of the siiccedaneous or fetus of 4 months hadl p)ogreSsedl to the permanent teeth. had progresse(l anywhere stage of deposition of enamel and (lentin from. a growing lamina to bud, cap, and bell matrix ill the cr1()wvlns of the luciduous teeth stages. Thus the various stages of tooth (le- and to l)eginning (leposition of enamel.-den- velopmlent to nearly complete amelogenesis tin maltrix ill the first permanent molar were observed. This 4-month fetus showed

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M-wommom ,'' )i V..- W. A Foi(. 6.- --Labeled stratum-intermedium cells (B) of monkey fetus. E, enamel matrix; 1, ameloblastL.; 8, . (ilematoxvlin stain.) Fe; 7. laleledl ccII aIlong outer enamnel epithelium of monkey fetus. .41 stellate reticulumi; B. connlcctive tissue. (Hem tox lin stain.)

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lic. 8.-Labeled cell in outer enamel epithelium (D) during advanced enamel-matrix deposition. Note absence of stellate reticulum cells. A, enamel matrix; B, ameloblasts; C, connective tissue. (Hematoxylin stain.) Ftc. 9.- ILabeled stratumi intermedium (A) and outer enamel epithelium (B) of monkey fetus. (Hemia- toxylin stain.) I ,ol. 44, No, 6 Il If! TO TIC POTENI'.T O1 ]'JJE EN]i.1IJEL ORGliN 12S9 histological evidence of beginning calcifiCa- Labeled cells were abundant in thle early tion in the m-iandibular first permanent mo- stages of the stratumn intermedium, when it lars, which confirms the observation. of was four to six cell layers thick. Labeling Swindler and Gavan,'3 who, from dissected also was seen frequently during ammelogene- specimens, have stated that calcification of sis, when one cell layer constituted the the permanent dentition begins prior to stratum intermedium andl these cells were birth in the rhesus monkey. oriented perpendicular to the ameloblast s The 4>-month-old baby monkey exhibit- (Figs. 5, 6, 9, 10). As the cells of the stratum cd permanent tooth germs from the cap intermedium lost this perpendicular orien- stage to enamel and dentin-matrix deposi- tation, only one occasional labeled cell Wtas tion. The second deciduous molars were not noted. These observations support Hunt erupted, but the crowns were fully formed. and Pavnter's" contention that Cohen's'7 The crowns of the first permanent molars were approaching completion but not erupt- ed. All the deciduous teeth except the second molars were erupted into the oral cavity, and the front teeth were in normal function. Tooth development could thus be followed from the dental lamina to the stage of func- tional occlusion when the slides from the fetus and the bahy monkey were studied corn bined. The importance of differential prolifera- tion in the process of morphogenesis of the enaiumel organ has been documented previ- ouslk: by morphologic studies14-16 and by tlritiated thyrmidine studies.'-4 Labeled cells wxere found in all layers of the enamel organ through the cap and early bell stage (Figs. 2 -5). At the late bell stage, the stellate retic- ulumcdid not exhibit further labeling (Figs. FwI. 10.-Labeled cells (B) in stratum inter- 7-10). Hunt and Paxvnter observed in guinea meidiumi at developing cusp tip of deciduous central pigs3 that the only labeled cells in the stel- incisor of monkey fetus. Labeled endothelial celi late ret iC ultii were along the stratum in- (A). (Heniatoxxliii stailn.) termiediumn at the junction of the outer and near the base of the hypothesis considering the redtuced-enamel. enamniel organ. Since the stellate reticulum epitheltium to be an inactive degenerating of the monkey did not exhibit any labeling, tissue is not correct. It appears that a pro- it mayte be considered a postmitotic-cell pop- liferative potential is present in the straitum- ulat ion. intermedtium laxer atnd the outer enamel 'Tlhere was a marked variation in labeling epithelial laxyer in the monkey during amelo- of the inner enamel epithelium in the late genesis, and these tissues could, therefore, bell stage during the formation of the enanm- pr-ovide protection to the underlying con- el matrix. Undifferentiated cells of the inner nective tissue if necessLry. enamel epithelium and preameloblasts ex- hibited labeling in all sections. Functioning Sunmnmary amneloblasts, however, never exhibited label- Mitotic activity within the enamel organs ing 1 h]our after thymidine injection. It ap- of developing teeth in a fetus and a b(1ay pearedl that both the cells of the stellate rhesuis monkey' was sttuied by tritiated thx - reticullum and the mature ameloblasts lost midine radioautography. their ability to divide as they became fll1V No labeling or _mitotic figures were found differentiated. The stellate reticulum dis- in the stellate reticulum. or in the function- appeared after the crown formation was ing ameloblasts. Labeled cells were observed complete, and the residual non-functioning in all the other layers of the enamel organ postaleloblsasts never showed labeling or during and following amelogenesis. It ap- divisionn figures. pears, therefore, that the reduced-enamel 1290 ENGLER, RAMFJORD, AND HINIKER J. dent. Res. November-December 1965 epithelium is not made up of degenerated 7. OWEN, M., and MACPHERSON, S. Cell Population Ki- netics of an Oskogenic Tissue. II. (USAEC Report cells as proposed by some investigators, BNL-6738, Brookhaven National Laboratory, 1963.) since a potential for proliferation was dem- 8. GREULICH, R. C. Epithelial DNA and RNA Syn- thetic Activities of the Gingival Margin, J. dent. Res., onstrated in these cells. 40:682, 1961 (abstract). 9. Cell Proliferation and Migration in the Epi- We would like to express our thanks to Miss thelial Attachment Collar of the Mouse Molar. (Ab- Joan E. Wilhelm, who prepared all the radioauto- stract 304, 40th General Meeting of the International graphs, and to Mr. Charles T. Knorpp, M.S., As- Association for Dental Research, 1962.) sistant Chief, Radioisotope Unit, for his technical 10. BEAGRIE, G. S., and SKOUGAARD, M. R. Observations Both on the Life Cycle of the Gingival Epithelial Cells of assistance. are from the Veterans Administra- Mice as Revealed by Autoradiography, Acta odont. tion Hospital, Ann Arbor, Michigan. scand., 20:15-31, 1962. 11. SKOUGAARD, M. R., and BEAGRIE, G. S. The Re- References newal of Gingival Epithelium in Marmosets (Calli- thrix jacchus) as Determined through Autoradiog- 1. HUNT, A. M., and PAYNTER, K. J. The Role of Cells raphy with Thymidine HI, Acta odont. scand., 20: in the Stratum Intermedium in the Development of 467-84, 1962. the Guinea Pig Molar: A Study of Cell Differentiation 12. DIMASSIMO, C. Proliferation and Migration of Cells and Migration Using Tritiated Thymidine, Arch. oral in the Gingival Epithelium, pp. iii, 58. Unpublished Blot., 8:65-78, 1963. M.S. thesis, Eastman Dental Dispensary, Rochester, 2. STARKEY, W. E. The Migration and Renewal of Trit- New York, 1963. ium Labeled Cells in the Developing Enamel Organ A. of of Rabbits, Brit. dent. J., 115:143-53, 1963. 13. SWINDLER, D. R., and GAVAN, J. Calcification 3. PAYNTER, K. J., and HUNT, A. M. Mitotic Activity the Mandibular Molars in Rhesus Monkeys, Arch. in Rat Molar Tooth Germs, J. dent. Res., 40:652, oral Biol., 7:727-34, 1962. 1961 (abstract). 14. SCHOUR, I., and MASSLER, M. Studies in Tooth De- 4. HWANG, W. S., TONNA, E. A., and CRONKITE, E. P. velopment: The Growth Pattern of Human Teeth. I, An Autoradiographic Analysis of the Cellular Growth J. Amer. dent. Ass., 27:1778-93, 1940. of Mouse Incisors Using Tritiated Thymidine. (Ab- 15. WAKEFORD, C. A. Some Observations on the Tooth stract 141 of the 41st General Meeting of the Inter- Band and on the Enamel Organs of the Human De- national Association for Dental Research, 1963.) ciduous Teeth, Proc. roy. Soc. Med. (Odontol. Scc.), 5. ENGLER, W. O., RAMFJoRD, S. P., and HINIKER, J. J. 20:13-28, 1927. The Development of the Epithelial Attachment and 16. LEFKOWITZ, W., BODECKER, C. F., and MARDFIN, the Gingival Sulcus in Rhesus Monkeys, J. Periodont. D. E. Odontogenesis of the Rat Molar: Prenatal (in press). Stage, J. dent. Res., 32:749, 1953 (abstract). 6. PELC, S. R. On the Question of Renewal of Differen- 17. COHEN, B. Pathology of Interdental Tissues, Dent. tiated Cells, Exp. Cell Res., 29:194-98, 1963. Practit. dent. Rec., 9:167-73, 1959.