aging of dental tissues remains at best, exiguous, despite our scientific sophisticaion and vast clinical experience which reveals that periodontal disease is one of the most widespread and chronic diseases of mankind, while its incidence and severity are known to increase with increasing a g e . A number of studies appear in print, including our own, which evaluate the magnitude of a variety of tissue parameters from one age to the other. Such studies are important and are a necessary first step in the establishment of base line information revealing changes in the status quo with increasing age. The data here largely reflect changes in physiological demands during the course of development, growth, remodeling and maintenance. However, they do not reveal the effects of age changes on the tissue potential to cope with inflammatory processes, surgical and accidental trauma, with the rate of response to these and other perturba tions or to the susceptibility of periodontal tissues. Such knowledge is germane to the understanding of the ef fects of age on a given system. To derive this kind of data, it is essential that periodontal tissue is studied under natural or induced stress throughout the life-span of the experimental subject. It has become apparent from earlier studies, ' of appendicular and axial bone, that with increasing age there occurs an accumulation of subtle, intrinsic, bio physical and biochemical changes which result in the progressively irreversible and deleterious alteration of basic cell mechanisms. Such are the parameters charac teristic of the aging syndrome. As the demands for growth diminish, accumulated age changes become more recognizable until the ensuing morphological changes are of such magnitude that age changes become unquestiona bly obvious. In the present description of age changes of dental bone and cementum, particular reference will be made to the Brookhaven National Laboratory Swiss albino (BNL) mouse, a short-lived inbred strain used extensively by the author as a mammalian model for aging of skeletal and dental tissues. The BNL mouse has a mean life-span of less than 1 year. Some animals can be bred to slightly over 2 years, but with great difficulty. Reference to dental tissues will be made to data derived from the parodontal tissues associated with the maxillary first molar. The parodontal tissues of this mouse reveal extensive age changes similar to those known to occur in the human. It must be pointed out that one must be cognizant of the differences which normally exist in the timing of similar specific events between rodents, man and other mammals, so that a proper compartive analy sis can be made. Utility of the BNL mouse as a model in the study of parodontal tissue aging is extended by the fact that the animal exhibits two natural inflammatory processes whose incidence and severity increase with age. A subcrevicular inflammation reveals an increase in inci dence of from 4.3% at 5 weeks to 55.5% at 78 weeks of age. Periapical inflammation is absent at 5 weeks, but
Factors (Aging) Affecting Bone and Cementum*
2
3,4
by EDGAR A . TONNA, M.S., PH.D., F.R.M.S.f of the structure and function of bone during growth, development, aging and in response to perturba tions has taught us that "the form of a bone being given, the bone elements place or displace themselves in the direction of functional forces and increase or decrease their mass to reflect the amount of functional forces." This statement was enunciated as far back as 1892 in Wolffs law, in which bone is seen to respond to the extrinsic forces or loads which it must support. This fundamental behavior of bone not only applies to axial and appendicular bone, but to all bone in general, including bone associated with dental tissues and the mineralized cementum. The very structure of bone is intimately linked to function, and the stability of the established structure is permanent only insofar as the qualitative and quantitative function of bone remains unchanged. Surely, the functional complexity of bone associated with dental tissues elicts significant variations in the rates at which cellular events occur and in cellular capacities. In addition, the superimposition of perturbations re sulting from inflammatory processes, malocclusion, loss of dentition, the existence of prosthetic devices, aging, etc., is translated into the exceptional structural form typical of dental bone, its behavior and propensi ties, in other words—its uniqueness. An attempt will be made in this presentation to briefly review what is generally considered as normal age concomitant changes in dental bone and cementum, and the effects of age on the response of these tissues to experimental injury. It must be pointed out that the cellular kinetic data which will be reviewed here has been obtained over the years in close collaboration with my colleague Dr. Sigmund S. Stahl. I would also like to present some of my recent unpublished findings regard ing the changes which occur in the turnover rates of bone at varying surfaces and cementum, the observed differ ent modes of bone behavior, as well as some electron microscopic changes which occur with increasing age in both of these tissues. It is unfortunate that the literature on the subject of THE STUDY
1
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* Presented at the 50th Anniversary of the Department of Periodontology of New York University College of Dentistry, on May 16-17, 1975, at the Plaza Hotel, New York, New York. Research was supported by N.I.C.H.D. grant #DE-03014 from the National Institutes of Health, Bethesda, Maryland † Institute for Dental Research, Dental Center of New York University, 339 East 25th St. New York, N. Y. 10010.
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increases to 73.3% of the population of 78-week-old mice. Although, the mouse has been used little in dental research because of its physical size, numerous advan tages exist over larger models. With the reported de velopment of simple oral surgical procedures, previous objections to its use have been obviated. 8
9
ALVEOLAR
BONE CHANGES
WITH A G E 10
It has been claimed by Gottlieb and Orban that with advancing age alveolar bone resorption (senile atrophy) occurs and may be related to gingival recession. In a study of 123 clinically healthy subjects ranging in age from 11 to 70 years, statistically significant alveolar bone resorption occurred while only an insignificant crest height reduction was observed. Recorded wide variations in measurements suggest the influence of other parameters. The existence of the alveolar process is believed to be dependent upon the presence of the den tition, since the loss of teeth is followed by gradual re sorption. Alveolar process resorption is known to occur, however without the former loss of dentition. Studies of the B N L mouse reveal extensive resorption of the alveolar crest and alveolar bone in general and no tooth loss. It appears more probable that tooth loss, inflammatory processes and other perturbations aug ment the process, as tooth loss augments mandibular osteoporosis. 11
12
8
Osteoporotic changes in the mandible increase with age, affecting some areas more than others as judged by
13
definitive variations in mineral density. Shortly after maturity, the jaw begins to show signs of involution, initially appearing in regions defined as growth resorp tion sites. Resorption contributing to remodeling dur ing growth persists, becoming more extensive with in creasing age. Such resorption when situated at specific sites along the alveolar crest, leads to the uneven distribution of bone density and the changes in shape characteristic of the edentulous senile mandible. The osteoporotic process recognized with increasing age is not dependent upon the presence or absence of teeth for its inception. Such changes appear to be a major cause of, rather than the result of tooth loss in the adult. In the BNL mouse the interradicular bone exhibits continu ous remodeling activity throughout its life-span, with extensive resorption of periosteal surfaces and endosteal channels leading in the old animal to a bony structure which appears to be osteoporotic. The marrow spaces of alveolar bone lined by endos teum also reveal age changes. In man the red marrow is replaced by fatty marrow. As the molars of aging mice exhibit mesial drift, similar to that which occurs in the human, resorption of the alveolar process continues at the mesial periodontal surface, resulting in the displace ment of endosteal structures into the periodontium. Bone apposition occurs at the periosteal surface juxtapo sition to the subgingival connective tissues. Conse quently, the alveolar bone becomes essentially free of vascular and marrow channels (Fig. 1), which is not the 14
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FIGURE 1. With increasing age, endosteum lined marrow channels of the alveolar bone become reduced in size. Mesial drift shifts such channels and vascular structures (arrows) towards the encroaching periodontium. Eventually, they are freed into the periosteum and the residual alveolar bone (B) becomes avascular and a channel free structure. ( C ) Cementum. 52-week-old BNL mouse. Hematoxylin stained H -proline autoradiograph. (Original magnification x 550.) 3
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case with interradicular bone. This combination, includ ing extensive crestal resorption leads to an alveolar bone structure which is significantly reduced in profile and solid. With increasing age, periosteal and endosteal cell numbers decrease per unit of surface exposed, and as such surfaces diminish with age. Significant morphologi cal and ultrastructural cell changes are observed in association with dental bone as reported for bone elsewhere. Age changes become significant in func tional cells as the organismic demands are reduced. Active surface osteogenic cells including osteogenic cell precursors (preosteoblasts) and osteoblasts are reduced to an inactive morphology resembling fibrocytes (Figs. 2 and 3). Endosteal cells generally reveal persistant activ ity, so that age changes are delayed, as at the interradicu lar region. Aging cells become fusiform and much 16-18
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reduced in size, exhibiting hyperchromatic nuclei. The cytoplasmic mass is also significantly reduced as mito chondrial numbers diminish, the Golgi complex is struc turally simplified, and the abundant rough endoplasmic reticulum indicative of matrical protein synthetic activity disappears, leaving behind ribosomes arranged in aggre gates or freely distributed. Matrix production eventually ceases. Degenerative changes appear in membranous structures, including swelling of mitochondrial cristae, vacuole formation, etc. Bone surfaces exhibit extensive osteoplastic activity, demarcated by the presence of an electron dense osmiophilic lamina as evidence of past surface microremodeling activity (Fig. 3). Osteocytes once formed are destined to age and eventually die. The earlier literature portrayed osteocytes as in a state of "limbo", wherein reactivation of cells via bone resorption occurs if the lacunar walls are opened.
FIGURE 2. The electron micrograph shows an active osteoblast of the periosteum (mesial surface) of alveolar bone in a 5-week-old mouse. Numerous mitochondria (m) and abundant rough endoplasmic reticulum (e) are observed in abundant cytoplasm. The functional nucleus exhibits little euchromatin distributed preferentially at the periphery of the nucleus. Mineralizing collagen matrix at the bone surface (arrow) (Original magnification x 16,900.)
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FIGURE 3. The electron micrograph shows significant changes in aging nonfunctional residual osteoblasts at the alveolar bone surface of a 52-week-old mouse. Cells have become fusiform; the nuclei (n) are hyperchromatic. The cytoplasm has been reduced with the loss of mitochondrial numbers, absence of rough endoplasmic reticulum and diminished Golgi complex. No osteoid layer is evident at the extensively microremodeled bone surface evidenced by electron dense multiple osmiophilic lamina (arrows). (Original magnification x 16,900.)
Certainly, this does not appear to be the case when such lacunae are opened via osteoclastic resorption; here the cells are destroyed in the process. Jande and B e l a n g e r , have described in young rats and chicks fine structural events which occur during the life cycle of an osteocyte. These changes represent in fact, physiological changes initially and age changes subse quently, which are augmented in progressively older animals. In aging alveolar bone of the BNL mouse similar changes are observed (Figs. 4, 5). Once osteocytes are formed matrix production continues to occur, so that initially the lacunae are absent and osteocytes resemble the osteoblasts from which they are derived. Subse quently, cytoplasmic and nuclear reduction occurs result ing in the formation of definitive lacunae. However, the osmiophilic lamina is absent and matrix production persists at a diminished rate. Nuclei become typically hyperchromatic as the euchromatin content appears to increase. Mitochondria diminish in number while the Golgi complex becomes smaller and simpler in structure. 19
20,21
18
The rough endoplasmic reticulum essentially disappears while free ribosomes become scattered in the diminished cytoplasm. Lysosomes appear, and degenerative changes involving swelling of ultrastructural components includ ing mitochondria, Golgi, residual endoplasmic reticulum, and the cytoplasmic ground substance are seen. The perinuclear space enlarges as the cells undergo lysis. Eventually, complete lysis occurs so that the lacunae are filled with the lysed fragments of osteocytes. The fact that alveolar osteocytes are seen to undergo degenerative age changes at any age is not to deny the capacity of alveolar bone osteocytes to participate in osteolysis, osteoplasis, protein synthesis, mucopolysaccharide regu lation or mineral homeostasis, for all of these activities can take place with varying degrees. Figure 6 reveals ample evidence of such activity, as repeated phased demands for either bone resorption or formation occur. Osteocytes consequently undergo active lacunar resorp tion (osteocytic-osteoclasis) and accretion (osteocyticosteplasis). Such activity was described in skeletal bone
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Aging Bone and Cementum 22,23
by a number of investigators, and in the aging BNL mouse as well. Autoradiographic s t u d i e s of rat and mouse using H -thymidine as a radiomarker show that the cellular proliferative activity of osteogenic cells is extremely low when compared with the activity of other parodontal tissues. In the rat, the labeling index is initially higher than in the mouse, but by 20 weeks postpartum the values become similar. This low level persists throughout the life-span of both animals. In the presence of inflamma tory processes, crestal labeling appears elevated in old animals. Alveolar bone is unique and unlike the normal situation observed in skeletal bone elsewhere, since an inflammatory process of one magnitude or another is often present juxtaposition to bone in human and experi mental animals alike. In response to gingival injury a reactivation of cell proliferation occurs at periosteal and endosteal surfaces. The magnitude depends on the physiological demands reaching such surfaces. When the cellular response of aging alveolar bone osteogenic tissue to trauma is compared some diminished activity and delay in response is observed at the alveolar crest and mesial alveolar bone surfaces (Fig. 7). The delayed response, 17
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unlike the diminished physiological activity is indicative of an irreversible, debilitating age change. With increas ing age, beyond 52 weeks in the BNL mouse, this feature is augmented leading to delayed repair of alveolar bone. At the periodontal surface of alveolar bone, a resorptive bone surface, response to distant trauma is minimal. Despite the reduced cell proliferative activity changes and delayed response to trauma observed in older animals, sufficient integrity of osteogenic cells exists to allow reactivation of cellular proliferation and the resupply of needed new functional progeny. In recently completed autoradiographic studies of dental bone matrix producing activity, alveolar, basal and interradicular bone surfaces were compared using H -proline as a radiomarker. The topographic method as developed for skeletal tissue, has been extended to the evaluation of dental bone, cementum and dentin. The method involves the multiple administration of radiolabel at prescribed time intervals. In the utilization of H -proline osteoblasts First incorporate the radiolabel. Over 90% of it is hydroxylated to HMiydroxyproline, an amino acid essential to the synthesis of matrical collagen. At active matrix-producing surfaces, the multiple label appears in autoradiographs in the form of discrete bands 3
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3
FIGURE 4. The electron micrograph shows an older osteocyte than that shown in Figure 5. It is found deeper in the alveolar bone of a 5-week-old mouse. Nuclear and cytoplasmic reduction has already occurred with significant loss of rough endoplasmic reticulum. The nucleus (n) appears hyperchromatic. A well formed lacuna (1) is evident. The perilacunar wall is lined by an osmiophilic lamina indicative of the absence of osteocyte bone matrix formation (arrows). (Original magnification x 21,125.)
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FIGURE 5. The electron micrograph shows the death of an old degenerating alveolar bone osteocyte undergoing extensive Swelling of organelles is evident (arrows). Bone matrix (M). 104-week-old BNL mouse. (Original magnification x 16,900.)
of silver grains (Fig. 8). With the progression of time and continued matrix apposition, the bands appear farther away from the formative surface. The investigator, therefore has at his disposal a system wherein accurate cellular uptake and turnover activities are revealed at the precise surfaces being evaluated. Evaluation here is made through cell grain counting versus time. Over the mat rices discrete bands can also be grain counted and further micrometric analysis furnishes band width measurements and distance each band appears to have moved from its formative surface in micrometers. Combining time with distance, an evaluation of weekly, daily or hourly rates of matrix production is obtained. In addition, topographic maps of all bone surfaces can be constructed which reveal the history of matrix production throughout the life-span of the organism. The method utilizes light microscopy, and routine paraffin thin sectioning of decalcified tissue, rather than the cumbersome grinding of thick undemineralized preparations and examination under fluorescence microscopy as is currently used in tetracycline methods. The topographic method exhibits a 10-fold increase in sensitivity, while cellular detail and information is preserved. These studies show the existence of two modes of dental bone surface activity. Mesial alveolar bone perios teal surfaces and endosteal surfaces show an initially high 29
lysis.
rate of matrix production and discrete banding (Fig. 9 A) . With increasing age, activity diminishes quickly then continues to diminish progressively so that band conflu ence is seen and reduced band movement occurs (Fig. 9 B) . Crestal activity is turned off before 26 weeks of age, whereas mesial alveolar bone surface activity persists throughout life, accounting for the continued mesial drift of the dentition. The periodontal surface of alveolar bone being a resorptive, rather than an appositional surface, is free of autoradiographic bands (Fig. 1). Basal bone, reveals the second mode of activity. The matrix-producing activity is initially high and discrete multiple bands appear. Decreased activity is observed with increasing age, however a second phase of activity reappears. A maximum rate, at times higher than the original value occurs at 52 weeks. This is followed by diminished rates for the remainder of the animal's life-span. Interradicular bone periosteal and endosteal surfaces exhibit both modes of bone forming activity (Fig. 10 A, B). The mesial and coronal periosteal surfaces show the basal bone type of activity, while the distal periosteal surface and interradicular endosteal surfaces reveal the first mode of activity observed in alveolar bone. Although the rate of matrix production for inter radicular endosteal surfaces is low in old animals, it represents the highest rate observed for all residually
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active surfaces. With increasing age all dental bone surface activities eventually diminish. Various surfaces are turned off at different time periods during the course of a life-time. Cellular morphological and ultrastructural age changes are augmented at these surfaces. At residually active surfaces, age changes are collectively delayed. CEMENTUM
CHANGES
WITH A G E
Cementum may be defined as a specialized, calcified tissue of mesodermal origin, a modified type of bone covering the anatomic root of the teeth. Both intrinsic and concomitant age changes are known to occur. A number of characteristic age changes are recognized, the etiology of which remains ill-defined. Unlike bone, cementum is not normally resorbed, and cemental depo sition appears to continue in both man and experimental animals throughout their life-span. Acellular cementum covers the root dentin from the cemento-enamel junction to the apex of the tooth, but is usually of a cellular type at the apical third. A similar acellular cemental deposition is seen along the interradicular surface. A linear relation ship between the thickness of cementum and age is reported from a study of 233 human teeth. In the rat, 31
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the rate of cementum apposition is greater in young animals. This is also true in the mouse; the rate decreases very rapidly after 5 weeks of age, and continues to decline more slowly with increasing age (Fig. 11 A, B). The total mass however increases at the root to a point of reducing the apical foramen, thereby augmenting the age changes to the pulp. It will be noted from the H -proline topographic study (Fig. 11 A), that autoradiographic banding is discrete and continuous with that of dentin at 5 weeks of age. N o bands are observed along periodontal and interradicular cemental surfaces at this time. In 26-week-old and older mice, labeling of cementum is minimal and no definitive bands are formed, yet large amounts of cementum accumulate (Fig. 12 B). The accumulation occurs at such a low rate that radiomarker uptake is near background and consequently, is not detected. If the rates of cementum formation were higher, undesirable hypercementosis would result. Addi tional evidence of this situation is seen in the continued deposition of acellular cementum at root apices in older mice. 33
8
3
Cementum also accumulates in large quantities along the interradicular surface of the tooth. In old mice the thickness of multilayered cementum is greater than the
FIGURE 6. The electron micrograph shows an alveolar bone osteocyte of a 52-week-old mouse. Note that the perilacunar walls exhibit multiple phases of bone resorption and apposition. The previous resorptive (osteocytic-osteoclastic) phase is scalloped and outlined by an osmiophilic lamina (large arrows). This is followed by an appositional phase (osteocytic-osteoplasis) currently in progress as the osmiophilic lamina is absent from the surface (small arrows). Lacuna (1). (Original magnification x 16,900.)
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FIGURE 7 A comparison is shown of the response of the osteogenic layer of alveolar bone to trauma (gingivectomy) in progressively older mice extending from the crest to the edentulous ridge at the maxillary first molar. Note the shift in peak time in the ^-thymidine derived percent labeling. The delayed response is indicative of an age change. (Copyright by American Dental Association. Reprinted by permission from Tonna, E. A. and Stahl, S. S. ). 26
FIGURE 8. An autoradiograph is shown of incisor dentin of a 5-week-old mouse previously treated with four time spaced doses of H -proline using the topographic method. Discrete bands of silver grains are formed. The arrows illustrate the distance measurements in tim for each band. Band width measurements and band grain counts can also be made. ( O ) Odontoblastic surface; ( P ) periodontal surface. (Original magnification x 550.) 3
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FIGURE 9. Topographic labeling of the mesial surface of alveolar bone of ( A ) a 5-week-old mouse and (B) a 52-week-old mouse are seen with four time spaced doses of H -proline. In A, four discrete bands are in evidence below the periosteum (arrows). Mucosal epithelium (e). Connective tissue (ct). In B, note that the four discrete bands have not appeared. Instead, a very thin layer of silver grains are seen juxtaposition with the formative surface (arrows). Connective tissue (ct). Hematoxylin stained. {Original magnification ( A ) x 550; (B) x 550.) 3
FIGURE 10. Interradicular bone at the maxillary first molar of mice ( A ) 5-week-old and (B) 104-week-old are seen topographically labeled with four time spaced doses of H -proline. In A , four discrete bands are formed (arrows). A thin layer of interradicular cementum is exhibited between the markers. Dentin (D). In B, evidence of extensive remodeling is present in the tortuous arrangement of subperiosteal reversal lines. Extensive remodeling and bone resorption continues throughout the animal's life span. Little bone matrix forming activity is detectable (arrows). Hematoxylin stained. (Original magnification ( A ) x 550; (B) x 550.) 3
thickness of tooth dentin (Compare Figs. 10 A and 12 A). Here, one sometimes encounters large focal deposition of cellular cementum (Fig. 12 B). The cellular incorporation at such sites may be indicative of the high rate of cementogenesis resulting in cellular trapping, normally observed at tooth apices. Continual reapposition of new
layers of cementum represents the aging of tooth as an organ, and maintains the attachment complex intact. The tooth is biologically as old as the youngest layer of cementum. Of course, this may be much less than its chronologic age. The deposition of cementum appears to occur in response to functional stress, but it must be 31
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FIGURE 11. Topographically labeled mesial root tips of the maxillary first molar of ( A ) 5-week-old and (B) 26-week-old mice are shown following four time spaced doses of H -proline. (D) Dentin and (C) cementum. Note that the cementum is cellular, however several empty lacunae (arrows) can be observed at this time. In B, band formation is minimal at the surface of cementum (arrows), and multiple bands are not formed. Cementocytes are hyperchromatic and pyknotic. Cementum (C). Numerous empty lacunae are in evidence. Hematoxylin stained. (Original magnification ( A ) x 550; (B) x 550.) 3
FIGURE 12. ( A ) The continuous accretion of interradicular cementum (between the arrow pairs) forms a layer thicker than the adjacent dentin (D) 104-week-old BNL mouse. Compare with Figure 10 A. In B, a coronal region of interradicular cementum (C) of a 52-week-old mouse reveals excessive focal deposition. The cellular cemental mass appears to have been formed at a great rate entrapping numerous cells (arrows). This event must have occurred prior to H -proline administration, since the interradicular periodontium is heavily labeled, whereas no labeling of cementum is observed. (D) dentin. Hematoxylin stained. (Original magnification ( A ) x 550; (B) x 550.) 3
pointed out that relatively thick layers of cementum are found at the roots of unerupted teeth of aged individuals. There exists evidence from polarization microscopy 12
studies of young rats, that for some distance from the cemento-enamel junction down, the thin layer of cemen tum consists of poorly mineralized cementoid. It is not known whether aging eliminates the cementoid layer, 34
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thereby reducing the resistance of cementum to resorp tion. Henry and Weinmann, have found that in the roots of 261 human teeth, areas of past and active resorption increased with increasing age. Similarly in aging mice, the frequency and severity of resorptive sites increase on the surface of the cementum extending below the cemento-enamel junction. Cementum appears to be more resistant to resorption than bone. This may be due to the presence of the cementoid covering seen over young cementum and possibly to the absence of vascular ity. Cementum therefore can withstand greater physical pressure than bone. With increasing age, vascularization of the cementum present at root apices is commonly observed. The significance of this observation is not clear. Extensive deposition of cementum (hypercementosis) involving part of a tooth, the whole tooth or all of the teeth also occurs with increasing age. If it improves the functional nature of the cementum, it is considered hypertrophy. Often excementoses (spurs) are formed leading to ankylosis with alveolar bone. Such events are also observed in the aging BNL mouse. Spurs are known to become detached forming cementicles in the periodon 35
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tal ligament. An increase in cementicle formation occurs in aging m a n , and mouse alike. Aging of acellular cementum is not readily discerned microscopically at either light or electron microscopic levels. Cellular cementum, however exhibits degeneration and death of cementocytes. Empty lacunae are eventually observed. The process of degeneration and death is apparently quite rapid, since a few empty lacunae can be seen in 5-week-old animals, while their numbers increase with age. Jande and Belanger, in an electron micro scopic study of young rat cementocytes reported a complete series of degenerative changes and death of cells. Once formed, cementocytes like osteocytes undergo similar changes leading finally to death, however the process of degeneration appears to be more rapid in cementocytes. The rapidity with which cementocyte degeneration and death occurs is perhaps due to a reduction in the accessibility of nutritive substances, and the successful elimination of cellular waste products. In a recently completed electron microscopic study of cementocytes in aging BNL mice, degenerative changes similar to those seen in young animals were observed in progressively older mice. The process does not appear to 31
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FIGURE 1 3 . The electron micrograph shows a degenerating cementocyte of a 52-week-old mouse. A condensed hyperchromatic nucleus is seen surrounded by scant cytoplasm free of most organelles. The well formed lacuna ( 1 ) exhibits its primary outline by the presence of a dense osmiophilic lamina (arrows). Subsequent to the cessation of cemental resorption, a phase of cementocytic-cementoplasis commenced, producing the narrowing of the lacuna (X). Matrix formation is in progress, since no osmiophilic lamina is present at the surface of the new matrix (X). (Original magnification x 16,900.)
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be enhanced with increasing age, since it is a rapid enough process in progress throughout the life-span of the animal. The events simply become more widespread. In young cellular cementum, surface layers examined with light microscopy reveal normal cementocytes resid ing in lacunae, as active cementoblasts continue to deposit cementum matrix at a rapid rate (Fig. 11 A). By 26 weeks, large numbers of empty lacunae are observed, while many of the cells exhibit hyperchromatic, pyknotic nuclei (Fig. 11 B). Beyond 26 weeks, one observes the cessation of cementocyte formation, so that acellular cementum is deposited. Degenerating residual cells and large number of empty lacunae are in evidence in Figure 12, representing a 104-week-old animal. To accomplish this, surface cementogenic cells remain viable through out the life of the animal. Electron microscopic observa tions reveal cellular age changes in such cells, but sig nificant deleterious changes are delayed, as they are in residually active interradicular endosteal cells. By com parison with cementoblasts, alveolar bone periosteal cells show extensive age changes. The distribution of cell degeneration and death in cellular cementum, as alluded to above, is unlike alveolar bone or bone elsewhere. Degeneration of osteocytes
occurs in the central portion of bone and viable cells are seen at the periphery. At the electron microscopic level it is seen that once formed, cementocytes initially resemble precursor ce mentoblasts. As described for bone, lacunae are initially absent. Almost immediately, cementocytes diminish in size; both the cytoplasm and nucleus become smaller. Acive cementocytic-cementoclasis occurs, which results in enlargement of the lacunae. This is often followed by active cementocytic-cementoplasis, where new matrix is deposited on the perilacunar walls resulting in a reduc tion of the size of the lacunae. Mineralization of the newly formed matrix subsequently occurs. When a given phase is terminated an osmiophilic lamina appears (Fig. 13). Sometimes phase reversal takes place so rapidly (resorption to apposition), that the osmophilic lamina is not formed. Size reduction in cementocytes initially involves loss of rough endoplasmic reticulum, mitochon drial numbers, and simplification of the Golgi complex. Ribosomes become scattered singly or in aggregates within the cytoplasm. Nuclei become further reduced in size and become hyperchromatic (Fig. 13). Lysosomes make their appearance. Subsequent degeneration of membranous structures of cells occurs as cell lysis 38
The electron micrograph shows the lysed remnants of a cementocyte of a 104-week-old mouse. Cellular debris (cd) fills the lacular space which was previously reduced by a phase of cementocytic-cementoplasis (X). The new matrix was laid down on the original perilacunar wall which is outlined by a highly irregular surfaced osmiophilic lamina (arrows). (Original magnification x 16,900.) FIGURE 1 4 .
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ensues. Mitochondria become swollen, and disruption of cristae occurs as the integrity of the cell and its plasma membrane is lost. All that remains is a "soup" of lysed cellular fragments (Fig. 14). The cellular proliferative activity of cementum is exceedingly low; being the lowest of all parodontal tissues. The labeling indices change little in rats and mice from youth to old age, although some progressive decrease with age is encountered, followed by a terminal rise in very old animals. The latter event occurs in all probability for the same reasons that exist for the terminal rise observed in gingival labeling indices. Despite the significantly low proliferative activity of cementogenic tissue and accumulating age changes, a proliferative response to trauma can be elicited in the oldest animals studied after a distant gingivectomy (Fig. 15) 2 7 , 3 9 , 4 0 52 weeks of age in the mouse, the response to trauma is diminished while peak activity is delayed to longer time periods. As alluded to earlier, the shift in peak time to longer periods is characteristic of an intrinsic age change. 24,25
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aging, trauma etc., is expected to be fundamentally similar to bone elsewhere. The forces which are charac teristic of the function of oral structures impart quantita tive rather than qualitative differences, which coupled with the element of time result in the unique form typical of dental bone. The existence of the two modes of dental bone surface activities are peculiar to the specific physio logical functions each surface must perform. Although physiological changes occur concomitantly with aging, intrinsic age changes are seen to accumulate with time in all bone cell types. Such changes are indeed progressive, irreversible and in some respects debilitating, in that the response of bone to trauma is not only diminished, but is delayed. Despite significant age changes, however, suffi cient integrity is retained by the residual population of aged periosteal and endosteal cells to ensure the orga nism with an adequate response to trauma throughout its life-span. In earlier reported studies of femoral bone, this property was found to reside in the highly active progeny of old cells. Following trauma the old cells were stimulated to divide, a property not lost by aging of bone cells. It is believed that a similar property is possessed by "old" dental bone cells, since such cells have been shown to be reversible postmitotics. 16
5
SUMMARY A N D CONCLUSIONS
Bone associated with the support of the dentition is basically similar to bone elsewhere. Consequently, its dynamic functional response to growth, development,
In many respects we have seen that cementum reveals significant similarities to bone in a number of parame-
3
A comparison of the H -thymidine determined percent labeling response of the cementogenic layer to a distant trauma (gingivectomy), in progressively older mice showing the typical age change detected in 52-week-old animals by shift in peak labeling time. (Copyright by American Dental Association. Reprinted by permission from Tonna, E. A. and Stahl, S. S. ). FIGURE 1 5 .
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ters, although specific differences also exist. It would appear from these studies that aside from the dissimilari ties, bone and cementum each exhibit significant age changes, each exhibit very low proliferative activity, but both retain the capacity to respond to renewed demands throughout the life of the organism. Aging appears to exert a limited debilitating effect on the response of these tissues; this is not to imply, however, that age changes are not significant to the increased incidence and severity of periodontal disease, since the aging of other paro dontal tissues including epithelial and soft connective tissues must also be considered. Simply stated, sufficient knowledge as to the effects of aging on the normal func tion of oral tissues and on rendering such tissues sus ceptible to periodontal disease is unavailable. Further more, the etiology of periodontal disease remains un known. Age changes coupled to other exogenous factors may yet be found to play a supporting role. REFERENCES
1. Wolff, J.: Das Gesetz der Transformation der Knochen. Hirschwald, Berlin, 1892. 2. Ramfjord, S. P., Emslie, R. D., Green, J. C , Held, A. J., and Waerhaug, J.: Epidemiological studies of periodontal disease. Am J Public Health 5 8 : 1713, 1968. 3. National Center for Health Statistics: Dental Findings in Adults by Age, Race, Sex, United States 1960-1962. Publica tion No. 1000, Series 11, No. 7, Government Printing Office. Washington, D.C. 4. Waerhaug, J.: Epidemiology of periodontal disease— review of the literature. Ramfjord, S. P., Kerr, D. A., and Ash, M. M., (eds), World Workshop in Periodontics, p 181, University of Michigan, Ann Arbor, 1966. 5. Tonna, E. A.: Skeletal cell aging and its effects on the osteogenetic potenial. Clin Orthop 4 0 : 57, 1965. 6. Tonna, E. A.: Response of the cellular phase of the skeleton to trauma. J Periodontol 4 : 105, 1966. 7. Tonna, E. A.: Parodontal inflammation and aging in the laboratory mouse. J Periodontol 4 3 : 403, 1972. 8. Tonna, E. A.: Histological age changes associated with mouse parodontal tissues. J Gerontol 2 8 : 1, 1973. 9. Tonna, E. A.: A routine mouse gingivectomy procedure for periodontal research in aging. J Periodont Res 7: 261, 1972. 10. Gottlieb, B., and Orban, B.: Dental Science and Dental Arts, S. M. Gordon (ed), London, H. Kimpton Pub., 1938. 11. Dean Boyle, W., Jr., Via, W. F., Jr., and McFall, W. T., Jr.: Radiographic analysis of alveolar crest height and age. J. Periodontol 4 4 : 236, 1973. 12. Miles, A. E. W.: Ageing in the Teeth and Oral Tissues. G. H. Bourne (ed), Structural Aspects of Ageing, p 352. New York, Hafner Pub., 1961. 13. Manson, J. D., and Lucas, R. B.: A microradiographic study of age changes in the human mandible. Arch Oral Biol 7: 761, 1962. 14. Enlow, D. H., and Harris, D. B.: A study of postnatal growth of the human mandible. Am J Orthod 5 0 : 25, 1964. 15. Atkinson, P. J., and Woodhead, C : Changes in human mandibular structure with age. Arch Oral Biol 13: 1453, 1968. 16. Tonna, E. A.: Electron microscopy of aging skeletal cells III. The periosteum. Lab Invest 3 1 : 609, 1974. 17. Tonna, E. A.: Electron microscopic evidence of alter nating osteocytic—osteoclastic and osteoplastic activity in
the perilacunar walls of aging mice. Conn Tissue Res 1: 221, 1972. 18. Tonna, E. A.: An electron microscopic study of skeletal cell aging—II. The osteocyte. Exp Gerontol 8 : 9, 1973. 19. Tonna, E. A.: An electron microscopic study of os teocyte release during osteoclasis in mice of different ages. Clin Orthop 8 7 : 311, 1972. 20. Jande, S. S., and Belanger, L. F.: Electron microscopy of osteocytes. and the pericellular matrix in rat trabecular bone. Calcif Tissue Res 6 : 280, 1971. 21. Jande, S. S.: Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chicks. J Ultrastruct Res 3 7 : 279, 1971. 22. Baud, C. A.: Morphologie et structure inframicroscopique des osteocytes. Acta Anat (Basel) 5 1 : 209, 1962. 23. Bélanger, L. F., and Migicovsky, B. B.: Histochemical evidence of proteolysis in bone: The influence of parathormone. J Histochem Cytochem 1 1 : 734, 1963. 24. Stahl, S. S., Tonna, E. A., and Weiss, R.: The effects of aging on the proliferative activity of rat periodontal structures. J Gerontol 2 4 : 447, 1969. 25. Tonna, E. A., Weiss, R., and Stahl, S. S.: The cell proliferative activity of parodontal tissues in aging mice. Arch Oral Biol 17: 969, 1972. 26. Tonna, E. A., and Stahl, S. S.: Comparative assessment of the cell proliferative activities of injured parodontal tissues in aging mouse. J Dent Res 5 3 : 609, 1974. 27. Tonna, E. A., Stahl, S. S., and Weiss, R.: Autoradio graphic evaluation of gingival response to injury. I I . Surgical trauma in young rats. Arch Oral Biol 14: 19, 1969. 28. Tonna, E. A.: Study of aging parodontal bone using the H -proline topographic method. Arch Oral Biol (in press), 1976. 29. Tonna, E. A.: Topographic labeling method using H-proline in assessment of skeletal growth and remodeling in 5-week-old mice. Lab Invest 3 0 : 161, 1974. 30. Tonna, E. A.: Application of the H -proline topographic method to dental tissues. J Periodont Res 10:357, 1975. 31. Kotanyi, E.: Cementum. H. Sicher (ed), Orban"s Oral Histology and Embryology, ed 6, p 155, St. Louis, Mo., C. V. Mosby Co., 1966. 32. Zander, H. A., and Hurzeler, B.: Continuous cementum apposition. J Dent Res 3 7 : 1035, 1958. 33. Louridis, O., Kyrkanidou, E. B., and Demetriou, N.: Age effect upon cementum width of albino rat: A histometric study. J Periodontol 4 3 : 533, 1972. 34. Tonna, E. A., and Stahl, S. S.: A polarized light microscopic study of rat periodontal ligament following surgi cal and chemical trauma. Helv Odontol Acta 11: 90, 1967. 35. Henry, J. L., and Weinmann, J. P.: The pattern of resorption and repair of human cementum. J Am Dent Assoc 4 2 : 270, 1951. 36. Jande, S. S., and Belanger, L. F.: Fine structural study of rat molar cementum. Anat Rec 167: 439, 1970. 37. Tonna, E. A.: An electron microscopic study of aging cementocytes. J Periodont Res (in press), 1976. 38. Tonna, E. A.: A study of osteocyte formation and distribution in aging mice complemented with H -proline autoradiography. J Gerontol 2 1 : 124, 1966. 39. Stahl, S. S., Tonna, E. A., and Weiss, R.: Autoradio graphic evaluation of gingival response to injury, I.Surgical trauma in young adult rats. Arch Oral Biol 1 3 : 71, 1968. 40. Tonna, E. A., and Stahl, S. S.: An H -thymidine autoradiographic study of cell proliferative activity on injured parodontal tissues of 5-week-old mice. Arch Oral Biol 18: 617, 1973. 3
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Factors (Aging) Affecting Bone and Cementum*
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by EDGAR A . TONNA, M.S., PH.D., F.R.M.S.f of the structure and function of bone during growth, development, aging and in response to perturba tions has taught us that "the form of a bone being given, the bone elements place or displace themselves in the direction of functional forces and increase or decrease their mass to reflect the amount of functional forces." This statement was enunciated as far back as 1892 in Wolffs law, in which bone is seen to respond to the extrinsic forces or loads which it must support. This fundamental behavior of bone not only applies to axial and appendicular bone, but to all bone in general, including bone associated with dental tissues and the mineralized cementum. The very structure of bone is intimately linked to function, and the stability of the established structure is permanent only insofar as the qualitative and quantitative function of bone remains unchanged. Surely, the functional complexity of bone associated with dental tissues elicts significant variations in the rates at which cellular events occur and in cellular capacities. In addition, the superimposition of perturbations re sulting from inflammatory processes, malocclusion, loss of dentition, the existence of prosthetic devices, aging, etc., is translated into the exceptional structural form typical of dental bone, its behavior and propensi ties, in other words—its uniqueness. An attempt will be made in this presentation to briefly review what is generally considered as normal age concomitant changes in dental bone and cementum, and the effects of age on the response of these tissues to experimental injury. It must be pointed out that the cellular kinetic data which will be reviewed here has been obtained over the years in close collaboration with my colleague Dr. Sigmund S. Stahl. I would also like to present some of my recent unpublished findings regard ing the changes which occur in the turnover rates of bone at varying surfaces and cementum, the observed differ ent modes of bone behavior, as well as some electron microscopic changes which occur with increasing age in both of these tissues. It is unfortunate that the literature on the subject of THE STUDY
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* Presented at the 50th Anniversary of the Department of Periodontology of New York University College of Dentistry, on May 16-17, 1975, at the Plaza Hotel, New York, New York. Research was supported by N.I.C.H.D. grant #DE-03014 from the National Institutes of Health, Bethesda, Maryland † Institute for Dental Research, Dental Center of New York University, 339 East 25th St. New York, N. Y. 10010.
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increases to 73.3% of the population of 78-week-old mice. Although, the mouse has been used little in dental research because of its physical size, numerous advan tages exist over larger models. With the reported de velopment of simple oral surgical procedures, previous objections to its use have been obviated. 8
9
ALVEOLAR
BONE CHANGES
WITH A G E 10
It has been claimed by Gottlieb and Orban that with advancing age alveolar bone resorption (senile atrophy) occurs and may be related to gingival recession. In a study of 123 clinically healthy subjects ranging in age from 11 to 70 years, statistically significant alveolar bone resorption occurred while only an insignificant crest height reduction was observed. Recorded wide variations in measurements suggest the influence of other parameters. The existence of the alveolar process is believed to be dependent upon the presence of the den tition, since the loss of teeth is followed by gradual re sorption. Alveolar process resorption is known to occur, however without the former loss of dentition. Studies of the B N L mouse reveal extensive resorption of the alveolar crest and alveolar bone in general and no tooth loss. It appears more probable that tooth loss, inflammatory processes and other perturbations aug ment the process, as tooth loss augments mandibular osteoporosis. 11
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Osteoporotic changes in the mandible increase with age, affecting some areas more than others as judged by
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definitive variations in mineral density. Shortly after maturity, the jaw begins to show signs of involution, initially appearing in regions defined as growth resorp tion sites. Resorption contributing to remodeling dur ing growth persists, becoming more extensive with in creasing age. Such resorption when situated at specific sites along the alveolar crest, leads to the uneven distribution of bone density and the changes in shape characteristic of the edentulous senile mandible. The osteoporotic process recognized with increasing age is not dependent upon the presence or absence of teeth for its inception. Such changes appear to be a major cause of, rather than the result of tooth loss in the adult. In the BNL mouse the interradicular bone exhibits continu ous remodeling activity throughout its life-span, with extensive resorption of periosteal surfaces and endosteal channels leading in the old animal to a bony structure which appears to be osteoporotic. The marrow spaces of alveolar bone lined by endos teum also reveal age changes. In man the red marrow is replaced by fatty marrow. As the molars of aging mice exhibit mesial drift, similar to that which occurs in the human, resorption of the alveolar process continues at the mesial periodontal surface, resulting in the displace ment of endosteal structures into the periodontium. Bone apposition occurs at the periosteal surface juxtapo sition to the subgingival connective tissues. Conse quently, the alveolar bone becomes essentially free of vascular and marrow channels (Fig. 1), which is not the 14
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FIGURE 1. With increasing age, endosteum lined marrow channels of the alveolar bone become reduced in size. Mesial drift shifts such channels and vascular structures (arrows) towards the encroaching periodontium. Eventually, they are freed into the periosteum and the residual alveolar bone (B) becomes avascular and a channel free structure. ( C ) Cementum. 52-week-old BNL mouse. Hematoxylin stained H -proline autoradiograph. (Original magnification x 550.) 3
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case with interradicular bone. This combination, includ ing extensive crestal resorption leads to an alveolar bone structure which is significantly reduced in profile and solid. With increasing age, periosteal and endosteal cell numbers decrease per unit of surface exposed, and as such surfaces diminish with age. Significant morphologi cal and ultrastructural cell changes are observed in association with dental bone as reported for bone elsewhere. Age changes become significant in func tional cells as the organismic demands are reduced. Active surface osteogenic cells including osteogenic cell precursors (preosteoblasts) and osteoblasts are reduced to an inactive morphology resembling fibrocytes (Figs. 2 and 3). Endosteal cells generally reveal persistant activ ity, so that age changes are delayed, as at the interradicu lar region. Aging cells become fusiform and much 16-18
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reduced in size, exhibiting hyperchromatic nuclei. The cytoplasmic mass is also significantly reduced as mito chondrial numbers diminish, the Golgi complex is struc turally simplified, and the abundant rough endoplasmic reticulum indicative of matrical protein synthetic activity disappears, leaving behind ribosomes arranged in aggre gates or freely distributed. Matrix production eventually ceases. Degenerative changes appear in membranous structures, including swelling of mitochondrial cristae, vacuole formation, etc. Bone surfaces exhibit extensive osteoplastic activity, demarcated by the presence of an electron dense osmiophilic lamina as evidence of past surface microremodeling activity (Fig. 3). Osteocytes once formed are destined to age and eventually die. The earlier literature portrayed osteocytes as in a state of "limbo", wherein reactivation of cells via bone resorption occurs if the lacunar walls are opened.
FIGURE 2. The electron micrograph shows an active osteoblast of the periosteum (mesial surface) of alveolar bone in a 5-week-old mouse. Numerous mitochondria (m) and abundant rough endoplasmic reticulum (e) are observed in abundant cytoplasm. The functional nucleus exhibits little euchromatin distributed preferentially at the periphery of the nucleus. Mineralizing collagen matrix at the bone surface (arrow) (Original magnification x 16,900.)
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FIGURE 3. The electron micrograph shows significant changes in aging nonfunctional residual osteoblasts at the alveolar bone surface of a 52-week-old mouse. Cells have become fusiform; the nuclei (n) are hyperchromatic. The cytoplasm has been reduced with the loss of mitochondrial numbers, absence of rough endoplasmic reticulum and diminished Golgi complex. No osteoid layer is evident at the extensively microremodeled bone surface evidenced by electron dense multiple osmiophilic lamina (arrows). (Original magnification x 16,900.)
Certainly, this does not appear to be the case when such lacunae are opened via osteoclastic resorption; here the cells are destroyed in the process. Jande and B e l a n g e r , have described in young rats and chicks fine structural events which occur during the life cycle of an osteocyte. These changes represent in fact, physiological changes initially and age changes subse quently, which are augmented in progressively older animals. In aging alveolar bone of the BNL mouse similar changes are observed (Figs. 4, 5). Once osteocytes are formed matrix production continues to occur, so that initially the lacunae are absent and osteocytes resemble the osteoblasts from which they are derived. Subse quently, cytoplasmic and nuclear reduction occurs result ing in the formation of definitive lacunae. However, the osmiophilic lamina is absent and matrix production persists at a diminished rate. Nuclei become typically hyperchromatic as the euchromatin content appears to increase. Mitochondria diminish in number while the Golgi complex becomes smaller and simpler in structure. 19
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The rough endoplasmic reticulum essentially disappears while free ribosomes become scattered in the diminished cytoplasm. Lysosomes appear, and degenerative changes involving swelling of ultrastructural components includ ing mitochondria, Golgi, residual endoplasmic reticulum, and the cytoplasmic ground substance are seen. The perinuclear space enlarges as the cells undergo lysis. Eventually, complete lysis occurs so that the lacunae are filled with the lysed fragments of osteocytes. The fact that alveolar osteocytes are seen to undergo degenerative age changes at any age is not to deny the capacity of alveolar bone osteocytes to participate in osteolysis, osteoplasis, protein synthesis, mucopolysaccharide regu lation or mineral homeostasis, for all of these activities can take place with varying degrees. Figure 6 reveals ample evidence of such activity, as repeated phased demands for either bone resorption or formation occur. Osteocytes consequently undergo active lacunar resorp tion (osteocytic-osteoclasis) and accretion (osteocyticosteplasis). Such activity was described in skeletal bone
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by a number of investigators, and in the aging BNL mouse as well. Autoradiographic s t u d i e s of rat and mouse using H -thymidine as a radiomarker show that the cellular proliferative activity of osteogenic cells is extremely low when compared with the activity of other parodontal tissues. In the rat, the labeling index is initially higher than in the mouse, but by 20 weeks postpartum the values become similar. This low level persists throughout the life-span of both animals. In the presence of inflamma tory processes, crestal labeling appears elevated in old animals. Alveolar bone is unique and unlike the normal situation observed in skeletal bone elsewhere, since an inflammatory process of one magnitude or another is often present juxtaposition to bone in human and experi mental animals alike. In response to gingival injury a reactivation of cell proliferation occurs at periosteal and endosteal surfaces. The magnitude depends on the physiological demands reaching such surfaces. When the cellular response of aging alveolar bone osteogenic tissue to trauma is compared some diminished activity and delay in response is observed at the alveolar crest and mesial alveolar bone surfaces (Fig. 7). The delayed response, 17
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unlike the diminished physiological activity is indicative of an irreversible, debilitating age change. With increas ing age, beyond 52 weeks in the BNL mouse, this feature is augmented leading to delayed repair of alveolar bone. At the periodontal surface of alveolar bone, a resorptive bone surface, response to distant trauma is minimal. Despite the reduced cell proliferative activity changes and delayed response to trauma observed in older animals, sufficient integrity of osteogenic cells exists to allow reactivation of cellular proliferation and the resupply of needed new functional progeny. In recently completed autoradiographic studies of dental bone matrix producing activity, alveolar, basal and interradicular bone surfaces were compared using H -proline as a radiomarker. The topographic method as developed for skeletal tissue, has been extended to the evaluation of dental bone, cementum and dentin. The method involves the multiple administration of radiolabel at prescribed time intervals. In the utilization of H -proline osteoblasts First incorporate the radiolabel. Over 90% of it is hydroxylated to HMiydroxyproline, an amino acid essential to the synthesis of matrical collagen. At active matrix-producing surfaces, the multiple label appears in autoradiographs in the form of discrete bands 3
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FIGURE 4. The electron micrograph shows an older osteocyte than that shown in Figure 5. It is found deeper in the alveolar bone of a 5-week-old mouse. Nuclear and cytoplasmic reduction has already occurred with significant loss of rough endoplasmic reticulum. The nucleus (n) appears hyperchromatic. A well formed lacuna (1) is evident. The perilacunar wall is lined by an osmiophilic lamina indicative of the absence of osteocyte bone matrix formation (arrows). (Original magnification x 21,125.)
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FIGURE 5. The electron micrograph shows the death of an old degenerating alveolar bone osteocyte undergoing extensive Swelling of organelles is evident (arrows). Bone matrix (M). 104-week-old BNL mouse. (Original magnification x 16,900.)
of silver grains (Fig. 8). With the progression of time and continued matrix apposition, the bands appear farther away from the formative surface. The investigator, therefore has at his disposal a system wherein accurate cellular uptake and turnover activities are revealed at the precise surfaces being evaluated. Evaluation here is made through cell grain counting versus time. Over the mat rices discrete bands can also be grain counted and further micrometric analysis furnishes band width measurements and distance each band appears to have moved from its formative surface in micrometers. Combining time with distance, an evaluation of weekly, daily or hourly rates of matrix production is obtained. In addition, topographic maps of all bone surfaces can be constructed which reveal the history of matrix production throughout the life-span of the organism. The method utilizes light microscopy, and routine paraffin thin sectioning of decalcified tissue, rather than the cumbersome grinding of thick undemineralized preparations and examination under fluorescence microscopy as is currently used in tetracycline methods. The topographic method exhibits a 10-fold increase in sensitivity, while cellular detail and information is preserved. These studies show the existence of two modes of dental bone surface activity. Mesial alveolar bone perios teal surfaces and endosteal surfaces show an initially high 29
lysis.
rate of matrix production and discrete banding (Fig. 9 A) . With increasing age, activity diminishes quickly then continues to diminish progressively so that band conflu ence is seen and reduced band movement occurs (Fig. 9 B) . Crestal activity is turned off before 26 weeks of age, whereas mesial alveolar bone surface activity persists throughout life, accounting for the continued mesial drift of the dentition. The periodontal surface of alveolar bone being a resorptive, rather than an appositional surface, is free of autoradiographic bands (Fig. 1). Basal bone, reveals the second mode of activity. The matrix-producing activity is initially high and discrete multiple bands appear. Decreased activity is observed with increasing age, however a second phase of activity reappears. A maximum rate, at times higher than the original value occurs at 52 weeks. This is followed by diminished rates for the remainder of the animal's life-span. Interradicular bone periosteal and endosteal surfaces exhibit both modes of bone forming activity (Fig. 10 A, B). The mesial and coronal periosteal surfaces show the basal bone type of activity, while the distal periosteal surface and interradicular endosteal surfaces reveal the first mode of activity observed in alveolar bone. Although the rate of matrix production for inter radicular endosteal surfaces is low in old animals, it represents the highest rate observed for all residually
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active surfaces. With increasing age all dental bone surface activities eventually diminish. Various surfaces are turned off at different time periods during the course of a life-time. Cellular morphological and ultrastructural age changes are augmented at these surfaces. At residually active surfaces, age changes are collectively delayed. CEMENTUM
CHANGES
WITH A G E
Cementum may be defined as a specialized, calcified tissue of mesodermal origin, a modified type of bone covering the anatomic root of the teeth. Both intrinsic and concomitant age changes are known to occur. A number of characteristic age changes are recognized, the etiology of which remains ill-defined. Unlike bone, cementum is not normally resorbed, and cemental depo sition appears to continue in both man and experimental animals throughout their life-span. Acellular cementum covers the root dentin from the cemento-enamel junction to the apex of the tooth, but is usually of a cellular type at the apical third. A similar acellular cemental deposition is seen along the interradicular surface. A linear relation ship between the thickness of cementum and age is reported from a study of 233 human teeth. In the rat, 31
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the rate of cementum apposition is greater in young animals. This is also true in the mouse; the rate decreases very rapidly after 5 weeks of age, and continues to decline more slowly with increasing age (Fig. 11 A, B). The total mass however increases at the root to a point of reducing the apical foramen, thereby augmenting the age changes to the pulp. It will be noted from the H -proline topographic study (Fig. 11 A), that autoradiographic banding is discrete and continuous with that of dentin at 5 weeks of age. N o bands are observed along periodontal and interradicular cemental surfaces at this time. In 26-week-old and older mice, labeling of cementum is minimal and no definitive bands are formed, yet large amounts of cementum accumulate (Fig. 12 B). The accumulation occurs at such a low rate that radiomarker uptake is near background and consequently, is not detected. If the rates of cementum formation were higher, undesirable hypercementosis would result. Addi tional evidence of this situation is seen in the continued deposition of acellular cementum at root apices in older mice. 33
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Cementum also accumulates in large quantities along the interradicular surface of the tooth. In old mice the thickness of multilayered cementum is greater than the
FIGURE 6. The electron micrograph shows an alveolar bone osteocyte of a 52-week-old mouse. Note that the perilacunar walls exhibit multiple phases of bone resorption and apposition. The previous resorptive (osteocytic-osteoclastic) phase is scalloped and outlined by an osmiophilic lamina (large arrows). This is followed by an appositional phase (osteocytic-osteoplasis) currently in progress as the osmiophilic lamina is absent from the surface (small arrows). Lacuna (1). (Original magnification x 16,900.)
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FIGURE 7 A comparison is shown of the response of the osteogenic layer of alveolar bone to trauma (gingivectomy) in progressively older mice extending from the crest to the edentulous ridge at the maxillary first molar. Note the shift in peak time in the ^-thymidine derived percent labeling. The delayed response is indicative of an age change. (Copyright by American Dental Association. Reprinted by permission from Tonna, E. A. and Stahl, S. S. ). 26
FIGURE 8. An autoradiograph is shown of incisor dentin of a 5-week-old mouse previously treated with four time spaced doses of H -proline using the topographic method. Discrete bands of silver grains are formed. The arrows illustrate the distance measurements in tim for each band. Band width measurements and band grain counts can also be made. ( O ) Odontoblastic surface; ( P ) periodontal surface. (Original magnification x 550.) 3
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FIGURE 9. Topographic labeling of the mesial surface of alveolar bone of ( A ) a 5-week-old mouse and (B) a 52-week-old mouse are seen with four time spaced doses of H -proline. In A, four discrete bands are in evidence below the periosteum (arrows). Mucosal epithelium (e). Connective tissue (ct). In B, note that the four discrete bands have not appeared. Instead, a very thin layer of silver grains are seen juxtaposition with the formative surface (arrows). Connective tissue (ct). Hematoxylin stained. {Original magnification ( A ) x 550; (B) x 550.) 3
FIGURE 10. Interradicular bone at the maxillary first molar of mice ( A ) 5-week-old and (B) 104-week-old are seen topographically labeled with four time spaced doses of H -proline. In A , four discrete bands are formed (arrows). A thin layer of interradicular cementum is exhibited between the markers. Dentin (D). In B, evidence of extensive remodeling is present in the tortuous arrangement of subperiosteal reversal lines. Extensive remodeling and bone resorption continues throughout the animal's life span. Little bone matrix forming activity is detectable (arrows). Hematoxylin stained. (Original magnification ( A ) x 550; (B) x 550.) 3
thickness of tooth dentin (Compare Figs. 10 A and 12 A). Here, one sometimes encounters large focal deposition of cellular cementum (Fig. 12 B). The cellular incorporation at such sites may be indicative of the high rate of cementogenesis resulting in cellular trapping, normally observed at tooth apices. Continual reapposition of new
layers of cementum represents the aging of tooth as an organ, and maintains the attachment complex intact. The tooth is biologically as old as the youngest layer of cementum. Of course, this may be much less than its chronologic age. The deposition of cementum appears to occur in response to functional stress, but it must be 31
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FIGURE 11. Topographically labeled mesial root tips of the maxillary first molar of ( A ) 5-week-old and (B) 26-week-old mice are shown following four time spaced doses of H -proline. (D) Dentin and (C) cementum. Note that the cementum is cellular, however several empty lacunae (arrows) can be observed at this time. In B, band formation is minimal at the surface of cementum (arrows), and multiple bands are not formed. Cementocytes are hyperchromatic and pyknotic. Cementum (C). Numerous empty lacunae are in evidence. Hematoxylin stained. (Original magnification ( A ) x 550; (B) x 550.) 3
FIGURE 12. ( A ) The continuous accretion of interradicular cementum (between the arrow pairs) forms a layer thicker than the adjacent dentin (D) 104-week-old BNL mouse. Compare with Figure 10 A. In B, a coronal region of interradicular cementum (C) of a 52-week-old mouse reveals excessive focal deposition. The cellular cemental mass appears to have been formed at a great rate entrapping numerous cells (arrows). This event must have occurred prior to H -proline administration, since the interradicular periodontium is heavily labeled, whereas no labeling of cementum is observed. (D) dentin. Hematoxylin stained. (Original magnification ( A ) x 550; (B) x 550.) 3
pointed out that relatively thick layers of cementum are found at the roots of unerupted teeth of aged individuals. There exists evidence from polarization microscopy 12
studies of young rats, that for some distance from the cemento-enamel junction down, the thin layer of cemen tum consists of poorly mineralized cementoid. It is not known whether aging eliminates the cementoid layer, 34
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thereby reducing the resistance of cementum to resorp tion. Henry and Weinmann, have found that in the roots of 261 human teeth, areas of past and active resorption increased with increasing age. Similarly in aging mice, the frequency and severity of resorptive sites increase on the surface of the cementum extending below the cemento-enamel junction. Cementum appears to be more resistant to resorption than bone. This may be due to the presence of the cementoid covering seen over young cementum and possibly to the absence of vascular ity. Cementum therefore can withstand greater physical pressure than bone. With increasing age, vascularization of the cementum present at root apices is commonly observed. The significance of this observation is not clear. Extensive deposition of cementum (hypercementosis) involving part of a tooth, the whole tooth or all of the teeth also occurs with increasing age. If it improves the functional nature of the cementum, it is considered hypertrophy. Often excementoses (spurs) are formed leading to ankylosis with alveolar bone. Such events are also observed in the aging BNL mouse. Spurs are known to become detached forming cementicles in the periodon 35
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tal ligament. An increase in cementicle formation occurs in aging m a n , and mouse alike. Aging of acellular cementum is not readily discerned microscopically at either light or electron microscopic levels. Cellular cementum, however exhibits degeneration and death of cementocytes. Empty lacunae are eventually observed. The process of degeneration and death is apparently quite rapid, since a few empty lacunae can be seen in 5-week-old animals, while their numbers increase with age. Jande and Belanger, in an electron micro scopic study of young rat cementocytes reported a complete series of degenerative changes and death of cells. Once formed, cementocytes like osteocytes undergo similar changes leading finally to death, however the process of degeneration appears to be more rapid in cementocytes. The rapidity with which cementocyte degeneration and death occurs is perhaps due to a reduction in the accessibility of nutritive substances, and the successful elimination of cellular waste products. In a recently completed electron microscopic study of cementocytes in aging BNL mice, degenerative changes similar to those seen in young animals were observed in progressively older mice. The process does not appear to 31
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FIGURE 1 3 . The electron micrograph shows a degenerating cementocyte of a 52-week-old mouse. A condensed hyperchromatic nucleus is seen surrounded by scant cytoplasm free of most organelles. The well formed lacuna ( 1 ) exhibits its primary outline by the presence of a dense osmiophilic lamina (arrows). Subsequent to the cessation of cemental resorption, a phase of cementocytic-cementoplasis commenced, producing the narrowing of the lacuna (X). Matrix formation is in progress, since no osmiophilic lamina is present at the surface of the new matrix (X). (Original magnification x 16,900.)
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be enhanced with increasing age, since it is a rapid enough process in progress throughout the life-span of the animal. The events simply become more widespread. In young cellular cementum, surface layers examined with light microscopy reveal normal cementocytes resid ing in lacunae, as active cementoblasts continue to deposit cementum matrix at a rapid rate (Fig. 11 A). By 26 weeks, large numbers of empty lacunae are observed, while many of the cells exhibit hyperchromatic, pyknotic nuclei (Fig. 11 B). Beyond 26 weeks, one observes the cessation of cementocyte formation, so that acellular cementum is deposited. Degenerating residual cells and large number of empty lacunae are in evidence in Figure 12, representing a 104-week-old animal. To accomplish this, surface cementogenic cells remain viable through out the life of the animal. Electron microscopic observa tions reveal cellular age changes in such cells, but sig nificant deleterious changes are delayed, as they are in residually active interradicular endosteal cells. By com parison with cementoblasts, alveolar bone periosteal cells show extensive age changes. The distribution of cell degeneration and death in cellular cementum, as alluded to above, is unlike alveolar bone or bone elsewhere. Degeneration of osteocytes
occurs in the central portion of bone and viable cells are seen at the periphery. At the electron microscopic level it is seen that once formed, cementocytes initially resemble precursor ce mentoblasts. As described for bone, lacunae are initially absent. Almost immediately, cementocytes diminish in size; both the cytoplasm and nucleus become smaller. Acive cementocytic-cementoclasis occurs, which results in enlargement of the lacunae. This is often followed by active cementocytic-cementoplasis, where new matrix is deposited on the perilacunar walls resulting in a reduc tion of the size of the lacunae. Mineralization of the newly formed matrix subsequently occurs. When a given phase is terminated an osmiophilic lamina appears (Fig. 13). Sometimes phase reversal takes place so rapidly (resorption to apposition), that the osmophilic lamina is not formed. Size reduction in cementocytes initially involves loss of rough endoplasmic reticulum, mitochon drial numbers, and simplification of the Golgi complex. Ribosomes become scattered singly or in aggregates within the cytoplasm. Nuclei become further reduced in size and become hyperchromatic (Fig. 13). Lysosomes make their appearance. Subsequent degeneration of membranous structures of cells occurs as cell lysis 38
The electron micrograph shows the lysed remnants of a cementocyte of a 104-week-old mouse. Cellular debris (cd) fills the lacular space which was previously reduced by a phase of cementocytic-cementoplasis (X). The new matrix was laid down on the original perilacunar wall which is outlined by a highly irregular surfaced osmiophilic lamina (arrows). (Original magnification x 16,900.) FIGURE 1 4 .
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Aging Bone and Cementum
ensues. Mitochondria become swollen, and disruption of cristae occurs as the integrity of the cell and its plasma membrane is lost. All that remains is a "soup" of lysed cellular fragments (Fig. 14). The cellular proliferative activity of cementum is exceedingly low; being the lowest of all parodontal tissues. The labeling indices change little in rats and mice from youth to old age, although some progressive decrease with age is encountered, followed by a terminal rise in very old animals. The latter event occurs in all probability for the same reasons that exist for the terminal rise observed in gingival labeling indices. Despite the significantly low proliferative activity of cementogenic tissue and accumulating age changes, a proliferative response to trauma can be elicited in the oldest animals studied after a distant gingivectomy (Fig. 15) 2 7 , 3 9 , 4 0 52 weeks of age in the mouse, the response to trauma is diminished while peak activity is delayed to longer time periods. As alluded to earlier, the shift in peak time to longer periods is characteristic of an intrinsic age change. 24,25
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aging, trauma etc., is expected to be fundamentally similar to bone elsewhere. The forces which are charac teristic of the function of oral structures impart quantita tive rather than qualitative differences, which coupled with the element of time result in the unique form typical of dental bone. The existence of the two modes of dental bone surface activities are peculiar to the specific physio logical functions each surface must perform. Although physiological changes occur concomitantly with aging, intrinsic age changes are seen to accumulate with time in all bone cell types. Such changes are indeed progressive, irreversible and in some respects debilitating, in that the response of bone to trauma is not only diminished, but is delayed. Despite significant age changes, however, suffi cient integrity is retained by the residual population of aged periosteal and endosteal cells to ensure the orga nism with an adequate response to trauma throughout its life-span. In earlier reported studies of femoral bone, this property was found to reside in the highly active progeny of old cells. Following trauma the old cells were stimulated to divide, a property not lost by aging of bone cells. It is believed that a similar property is possessed by "old" dental bone cells, since such cells have been shown to be reversible postmitotics. 16
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SUMMARY A N D CONCLUSIONS
Bone associated with the support of the dentition is basically similar to bone elsewhere. Consequently, its dynamic functional response to growth, development,
In many respects we have seen that cementum reveals significant similarities to bone in a number of parame-
3
A comparison of the H -thymidine determined percent labeling response of the cementogenic layer to a distant trauma (gingivectomy), in progressively older mice showing the typical age change detected in 52-week-old animals by shift in peak labeling time. (Copyright by American Dental Association. Reprinted by permission from Tonna, E. A. and Stahl, S. S. ). FIGURE 1 5 .
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ters, although specific differences also exist. It would appear from these studies that aside from the dissimilari ties, bone and cementum each exhibit significant age changes, each exhibit very low proliferative activity, but both retain the capacity to respond to renewed demands throughout the life of the organism. Aging appears to exert a limited debilitating effect on the response of these tissues; this is not to imply, however, that age changes are not significant to the increased incidence and severity of periodontal disease, since the aging of other paro dontal tissues including epithelial and soft connective tissues must also be considered. Simply stated, sufficient knowledge as to the effects of aging on the normal func tion of oral tissues and on rendering such tissues sus ceptible to periodontal disease is unavailable. Further more, the etiology of periodontal disease remains un known. Age changes coupled to other exogenous factors may yet be found to play a supporting role. REFERENCES
1. Wolff, J.: Das Gesetz der Transformation der Knochen. Hirschwald, Berlin, 1892. 2. Ramfjord, S. P., Emslie, R. D., Green, J. C , Held, A. J., and Waerhaug, J.: Epidemiological studies of periodontal disease. Am J Public Health 5 8 : 1713, 1968. 3. National Center for Health Statistics: Dental Findings in Adults by Age, Race, Sex, United States 1960-1962. Publica tion No. 1000, Series 11, No. 7, Government Printing Office. Washington, D.C. 4. Waerhaug, J.: Epidemiology of periodontal disease— review of the literature. Ramfjord, S. P., Kerr, D. A., and Ash, M. M., (eds), World Workshop in Periodontics, p 181, University of Michigan, Ann Arbor, 1966. 5. Tonna, E. A.: Skeletal cell aging and its effects on the osteogenetic potenial. Clin Orthop 4 0 : 57, 1965. 6. Tonna, E. A.: Response of the cellular phase of the skeleton to trauma. J Periodontol 4 : 105, 1966. 7. Tonna, E. A.: Parodontal inflammation and aging in the laboratory mouse. J Periodontol 4 3 : 403, 1972. 8. Tonna, E. A.: Histological age changes associated with mouse parodontal tissues. J Gerontol 2 8 : 1, 1973. 9. Tonna, E. A.: A routine mouse gingivectomy procedure for periodontal research in aging. J Periodont Res 7: 261, 1972. 10. Gottlieb, B., and Orban, B.: Dental Science and Dental Arts, S. M. Gordon (ed), London, H. Kimpton Pub., 1938. 11. Dean Boyle, W., Jr., Via, W. F., Jr., and McFall, W. T., Jr.: Radiographic analysis of alveolar crest height and age. J. Periodontol 4 4 : 236, 1973. 12. Miles, A. E. W.: Ageing in the Teeth and Oral Tissues. G. H. Bourne (ed), Structural Aspects of Ageing, p 352. New York, Hafner Pub., 1961. 13. Manson, J. D., and Lucas, R. B.: A microradiographic study of age changes in the human mandible. Arch Oral Biol 7: 761, 1962. 14. Enlow, D. H., and Harris, D. B.: A study of postnatal growth of the human mandible. Am J Orthod 5 0 : 25, 1964. 15. Atkinson, P. J., and Woodhead, C : Changes in human mandibular structure with age. Arch Oral Biol 13: 1453, 1968. 16. Tonna, E. A.: Electron microscopy of aging skeletal cells III. The periosteum. Lab Invest 3 1 : 609, 1974. 17. Tonna, E. A.: Electron microscopic evidence of alter nating osteocytic—osteoclastic and osteoplastic activity in
the perilacunar walls of aging mice. Conn Tissue Res 1: 221, 1972. 18. Tonna, E. A.: An electron microscopic study of skeletal cell aging—II. The osteocyte. Exp Gerontol 8 : 9, 1973. 19. Tonna, E. A.: An electron microscopic study of os teocyte release during osteoclasis in mice of different ages. Clin Orthop 8 7 : 311, 1972. 20. Jande, S. S., and Belanger, L. F.: Electron microscopy of osteocytes. and the pericellular matrix in rat trabecular bone. Calcif Tissue Res 6 : 280, 1971. 21. Jande, S. S.: Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chicks. J Ultrastruct Res 3 7 : 279, 1971. 22. Baud, C. A.: Morphologie et structure inframicroscopique des osteocytes. Acta Anat (Basel) 5 1 : 209, 1962. 23. Bélanger, L. F., and Migicovsky, B. B.: Histochemical evidence of proteolysis in bone: The influence of parathormone. J Histochem Cytochem 1 1 : 734, 1963. 24. Stahl, S. S., Tonna, E. A., and Weiss, R.: The effects of aging on the proliferative activity of rat periodontal structures. J Gerontol 2 4 : 447, 1969. 25. Tonna, E. A., Weiss, R., and Stahl, S. S.: The cell proliferative activity of parodontal tissues in aging mice. Arch Oral Biol 17: 969, 1972. 26. Tonna, E. A., and Stahl, S. S.: Comparative assessment of the cell proliferative activities of injured parodontal tissues in aging mouse. J Dent Res 5 3 : 609, 1974. 27. Tonna, E. A., Stahl, S. S., and Weiss, R.: Autoradio graphic evaluation of gingival response to injury. I I . Surgical trauma in young rats. Arch Oral Biol 14: 19, 1969. 28. Tonna, E. A.: Study of aging parodontal bone using the H -proline topographic method. Arch Oral Biol (in press), 1976. 29. Tonna, E. A.: Topographic labeling method using H-proline in assessment of skeletal growth and remodeling in 5-week-old mice. Lab Invest 3 0 : 161, 1974. 30. Tonna, E. A.: Application of the H -proline topographic method to dental tissues. J Periodont Res 10:357, 1975. 31. Kotanyi, E.: Cementum. H. Sicher (ed), Orban"s Oral Histology and Embryology, ed 6, p 155, St. Louis, Mo., C. V. Mosby Co., 1966. 32. Zander, H. A., and Hurzeler, B.: Continuous cementum apposition. J Dent Res 3 7 : 1035, 1958. 33. Louridis, O., Kyrkanidou, E. B., and Demetriou, N.: Age effect upon cementum width of albino rat: A histometric study. J Periodontol 4 3 : 533, 1972. 34. Tonna, E. A., and Stahl, S. S.: A polarized light microscopic study of rat periodontal ligament following surgi cal and chemical trauma. Helv Odontol Acta 11: 90, 1967. 35. Henry, J. L., and Weinmann, J. P.: The pattern of resorption and repair of human cementum. J Am Dent Assoc 4 2 : 270, 1951. 36. Jande, S. S., and Belanger, L. F.: Fine structural study of rat molar cementum. Anat Rec 167: 439, 1970. 37. Tonna, E. A.: An electron microscopic study of aging cementocytes. J Periodont Res (in press), 1976. 38. Tonna, E. A.: A study of osteocyte formation and distribution in aging mice complemented with H -proline autoradiography. J Gerontol 2 1 : 124, 1966. 39. Stahl, S. S., Tonna, E. A., and Weiss, R.: Autoradio graphic evaluation of gingival response to injury, I.Surgical trauma in young adult rats. Arch Oral Biol 1 3 : 71, 1968. 40. Tonna, E. A., and Stahl, S. S.: An H -thymidine autoradiographic study of cell proliferative activity on injured parodontal tissues of 5-week-old mice. Arch Oral Biol 18: 617, 1973. 3
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