Light and scanning electron microscopic observations of the canalicular system in human cellular cementum.код для вставкиСкачать
THE ANATOMICAL RECORD 222:121-127 (1988) Light and Scanning Electron Microscopic Observations of the Canalicular System in Human Cellular Cementum Department of OSAMA S. ISMAIL AND DENNIS F. WEBER Histology, University of Illinois, College of Dentistry, 801 South Paulina, Chicago, Illinois 60612 ABSTRACT A scanning electron microscopic cast technique was used to determine the nature of the canalicular system in human cellular cementum. Prior light microscopicobservations suggested the presence of two distinct types of lacunae: bonelike and a large irregular type generally confined to the interradicular region. Only the bone-like lacunae were visualized in the SEM cast preparations. The canalicular system associated with the bone-like lacunae was usually continuous from the surface of the dentin to the surface of the cementum in newly-erupted teeth. Casts having a sponge-like configuration were observed near the cementodentinal junction in some of the specimens from the interradicular region. The presence of these casts could not be predicted from prior light microscopic observations and it was concluded that they may represent infiltration of hypomineralized matrix rather than lacunae. Previous transmission electron microscopic (TEM) studies have shown that the more deeply-positioned cells in cellular cementum in man (e.g., Furseth, 1967, 1969)and other species (e.g., Jande and Belanger, 1970; Furseth, 1970, 1977) appear to be dead or moribund. One explanation for this apparent morbidity is the canalicular mechanism’s lack of enough efficiency to maintain the vitality of cells when, as a result of continued appositional growth of the tissue, they come to lie at progressively greater distances from the periodontal vasculature. Other reasons for the observed cellular morbidity must also be considered. Cellular cementum is a difficult tissue to fix. It is essentially avascular and the cells are accessible to the fixative only via minute canals (the canaliculi) having a diameter of a micrometer or less. The rate of diffusion of the fixative through these channels may not be rapid enough to adequately fur the more deeply-positioned cells. This apparent morbidity may therefore be the result of poor fixation rather than cell death. The nature of the canalicular network must also be considered. Is there a continuous canalicular network interconnecting the lacunae throughout the full thickness of cellular cementum? Classical light microscopic and TEM methods cannot unequivocally answer this question. Light microscopic observations show that the canaliculi in cellular cementum tend to radiate toward the source of nutrition, the periodontal ligament, and often present the illusion that the lacunae and canaliculi are isolated units. Vital cells, of course, could not be maintained in isolated lacunar units. The purpose of this present investigation was to determine to what extent, if any, the lacunae in cellular cementum are interconnected, both with each other and ultimately with the surface of the cementum. A scanning electron microscopic (SEM) cast technique (Weber, 1983; Curtis et al., 1985) which permits the 1988 ALAN R. LISS, mc. three-dimensional visualization of internal spaces in hard tissues was used to examine the canalicular system in human cellular cementum. MATERIALS AND METHODS Fifty non-carious, newly-erupted human third molars obtained from the University of Illinois Oral Surgery Clinic were used in the present investigation. A saw microtome employing an annular water-cooled diamond blade was used to obtain 2 to 3 300pm mesiodistal longitudinal sections from the central region of each tooth. These sections were examined light microscopically to determine the location and the amount of cellular cementum present. Although the amount of cellular cementum present varied considerably, all of the teeth had at least some cellular cementum present in the interradicular regions. A heavy gauge razor blade was used to cut small (approx. lmm x 1.5mm) segments from selected areas of the root. These segments contained the full thickness of the cementum along with a thin layer of the underlying dentin. The interradicular cellular cementum often contained many large irregular lacunae, whereas the lacunae seen in other regions were more similar in size and configuration to those seen in bone. Therefore, segments taken from the interradicular regions were separated from those taken elsewhere. The segments were infiltrated with methyl methacrylate monomer by either vacuum or replacement infiltration as previously described (Weber, 1983). After polymerization under UV light in 16mm flatbottom glass vials, the segments were reduced to 3080pm on plastic microscope slides using an integrated Received September 29, 1987; accepted January 28, 1988. Address reprint requests to Dr. Dennis F. Weber, Department of Histology, University of Illinois, College of Dentistry, 801 South Paulina, Chicago, IL 60612. 122 O.S. ISMAIL AND D.F. WEBER mounting and reduction technique (Weber et al., 1982). Photomicrographs of selected areas were taken with ordinary transmitted light. The areas photographed were scored with a razor blade so that light and SEM image$ could be compared. For SEM processing, a lOmm x lOmm segment containing the specimen was cut out of the plastic microscope slide with a revolving diamond disk. Complete matrix removal cast preparations were processed for SEM observations. These were produced as previously described in our studies of the canalicular system in human cortical bone (Curtis et al., 1985). The light microscopic image of the initial saw microtome-cut sections differed from that of the same section after methacrylate embedment and further reduction in thickness. The lacunae and canaliculi in the uninfiltrated thicker sections were readily visible as darkened structures, whereas in the thinner embedded sections, many (but not all) of the lacunae were translucent and the canaliculi of these lacunae were not visible at all. A variable number of lacunae, however, often retained a darkened appearance, suggesting that these lacunae represent isolated units which do not become infiltrated with the resin because their canaliculi are not part of a continuous system. A different series of light microscopic observations were made to determine if this apparent absence of infiltration was a peculiarity of the resin embedding and reduction technique. Additional saw-microtome sections were cut at 200pm as previously described and dried overnight in a vacuum desiccator. Selected areas containing a high density of lacunae were photographed without using mounting medium or cover slips. These sections were then slowly infiltrated with mounting medium by first placing the specimens in xylol and gradually adding increasing amounts of a synthetic mounting media over a twenty-four hour period. A cover slip was then placed over the specimen and the same area rephotographed. Some of these specimens were then embedded in methyl methacrylate to produce SEM cast preparations. opaque in light microscopic observations (Fig. 51, no lacunar casts were observed in the SEM preparations even though the underlying dentinal tubules were obviously well-infiltrated with the resin (Fig. 6). In sections in which most of the lacunae became translucent after resin embedding, an extensive canalicular cast system was observed. Lacunae-to-lacunae canalicular connections appeared to form a continuous network throughout the full thickness of the cellular cementum (Figs. 7-10). The lacunae, particularly in the thicker sections (e.g., Fig. 71, were often difficult to visualize because of the surrounding superimposed dense network formed by the canalicular system. Furthermore, the canalicular network did not appear t o be formed from a one-to-one direct connection between canaliculi of adjacent lacunae, as seen in lamellar bone. Instead, the canalicular system appeared to have a complicated branching system which had a reticular configuration (Fig. 9). Casts having a sponge-like configuration were occasionally observed in the interradicular regions near the cemento-dentinal junction (Fig. 10). In low magnification scanning electron micrographs, these casts appeared to be individual, interconnected units which were much larger than the bone-like lacunar casts (Fig. 10). Prior light microscopic observations of the sections containing these casts did not suggest their presence. OBSERVATIONS Fig. 2. Ordinary light photomicrograph of the area shown in Fig. 1 after infiltration of the specimen with mounting media. The bonelike lacunae have become translucent (arrows) and are difficult to visualize. Canaliculi are no longer visible; x 142. As observed with the light microscope, the lacunae and canaliculi in cellular cementum were readily visible as opaque structures in unmounted dried sections (Fig. 1).They appeared to be similar in size to those observed in bone. In most of the specimens, all of the lacunae became translucent and the canaliculi were no longer visible after thorough infiltration with mounting medium (Fig. 2). In some specimens, however, varying numbers of lacunae remained as opaque structures despite repeated efforts to infiltrate the specimen with mounting media (Fig. 3). Larger irregularly-shaped lacunae were often observed in the interradicular dentin. These lacunae were usually elongate in configuration, with their long axes oriented generally perpendicular to the surface of the cementum (Fig. 4). As with the bone-like lacunae, these lacunae often became translucent (not shown) after infiltration with mounting media. No casts corresponding in size and configuration to these lacunae, if they are indeed lacunae, were seen in subsequent SEM observations. As with mounting media infiltration, the light microscopic image of the lacunae varied in the methacrylate-embeddedspecimens. When the lacunae appeared DISCUSSION The light microscopic and SEM observations in the present investigation show that the lacunar-canalicular system in human cementum is quite heterogeneous in Fig. 1. Ordinary light photomicrograph of a thick (100-um) air-dned ground section of cellular cementum from the interrahcular region of a human molar tooth. Outer surface of the cementum can be seen in the left-hand portion of the field. Arrows indicate cemento-dentinal junction. Many dark, bonelike lacunae can be seen distributed throughout the thickness of the cellular cementum; x 142. Fig. 3. Ordinary light photomicrograph of a ground section of cellular cementum that was infiitrated with mounting media. Many bonelike lacunae have become translucent (small arrow); others remain dark and retain the appearance seen in uninfidtrated specimens. Canaliculi can be observed in association with the dark lacunae; x 142. Fig. 4. Ordinary light photomicrograph of an Aral&te-infiltratedground section of cellular cementum taken from the interradicular region. A number of large, dark, elongated lacunae can be seen. When this specimen was prepared for scanning electron microscopy, no casts corresponding to these lacunae could be observed; ~ 6 8 . Fig. 5. Ordinary light photomicrograph of a methyl methacrylateinfiltrated ground section of cellular cementum ( C ) and a portion of the underlying dentin (I)). Many dark, bonelike lacunae and canaliculi can be seen distributed throughout the full thickness of the cementum. Open arrow indicates the cemento-dentinaljunction; X 72. Fig. 6. Scanning electron micrograph of the preparation shown in Fig. 5. With the exception of one small area in the upper left portion of the field, no casts of lacunae or canaliculi can be seen in the region previously occupied by the cellular cementum. Dentinal tubules (T) have been infiltrated. They do not, however, extend to the cemento-dentinal junction (open arrow), probably because the outermost portion of the radicular dentine had undergone sclerosis. x 72. 126 O.S. ISMAIL AND D.F. WEBER a number of respects. The size and shape of the lacunae vary and the number of lacunae per unit volume also vary. The canalicular system was found to vary, from a continuous network (extending from the surface of the dentin to the surface of the cementum) to totally isolated lacunar-canalicular units. Vital cells cannot be maintained in the latter arrangement, but the potential for maintaining vital cells in a continuous system clearly exists. Since the most frequently observed lacunarcanalicular system had bone-like lacunae interconnected by a continuous canalicular system, we suggest that the ultrastructure of the more deeply-positioned cells in cellular cementum should be re-evaluated. As was mentioned in the Introduction, most authors have suggested that these deeper cells are moribund. As has been suggested for bone (Matthews, 1980), it may be that the degenerative appearance of these cells is the result of poor fixation rather than cell morbidity. In order for a fixative to reach the cells in a mineralized tissue such as celluar cementum or bone, the fixative must diffuse through a series of minute channels over a distance which can be quite substantial when considering the distance that the deepest cells lie from the vascular elements. In previous studies of human cellular cementum, the intact tissue was fixed by immersion and therefore the only surface of the tissue exposed to the fixative was the outer surface of the cementum. Perfusion fixation has been used in studies of non-hu- Fig. 7. Split screen scanning electron microgrpah of the specimens shown in Figs. 1and 2. After viewing the specimen with the light microscope as previously described, the specimen was reembedded in methyl methacrylate and prepared as a cast preparation. The low-power view on the lee includes the entire thickness of the cellular cementum. An extensive and apparently continuous canalicular cast system extends throughout the full thickness of the cementum in this relatively thick preparation; x 206. Higher magmilcation of the framed area seen in the right hand portion of the field illustrates even more dramatically the extensive nature of the canalicular system, which tends to obscure most of the lacunae (arrows).Background is cast system of underlying lacunae and canaliculi; x 1,069. Fig. 8. Split screen scanning electron micrograph of a cast preparation. The low-power view on the left shows a segment of cellular cementum taken from the apical region of a human molar. A portion of another segment can be seen in the upper righthand portion of the field. An extensive cast system can be seen throughout the full thickness of apical segment; x 35. A higher magmtkation of the framed area is shown on the right. Lacunae (arrows) are more readily visible in this thin preparation. Dark background represents surface of the plastic microscope slide on which the specimen was prepared, x 357. Fig. 9. Split-frame scanning electron micrograph taken from a different area of the specimen shown in Fig. 8. The lower-power view on the top is taken from the central one-third of the cellular cementum. The canalicular system appears to be continuous throughout the field; x 189. The lower portion of the field is a higher magnification of the framed area: one lacuna (L) is visible in the lower portion of the field, and an extensive branching canalicular network can be seen throughout the field x2197. Fig. 10. Split-screen scanning electron micrograph; the upper one-half of the field is a lower-power view of a portion of a segment taken from the interradicular region of a human molar. An extensive cast system can be seen throughout the full thickness of the cellular cementum, and casts of the tubules can be seen in the underlying dentin (D).The casts in the framed area near the cernento-dentinal junction appear to be larger than those seen elsewhere in the cementum; x 45. The lower onehalf of the field is a higher magnification view of the framed area. An extensive cast system with a spongelike appearance can be seen within the field; x 198. man cellular cementum. Although this method is generally superior t o immersion fixation, it would appear to be only minimally superior for the fixation of hard tissues because the fmative must still diffuse through the canalicular system to reach deeply-positioned cells. It is suggested, therefore, that optimal fixation of human cellular cementum would be enhanced by rapidly sectioning the tissue immediately prior to initial exposure to the fixative. Such a technique has been successfully employed to fix the contents of the dentinal tubules in human coronal dentine (Gaynor et al., 1984). Sections of cementum in the range of 50-150 um can be easily cut with a saw microtome using a water-cooled diamond-edged blade. Although some of the cells along the cut surface may be destroyed and heat damage may occur along this surface, many cells within the bulk of the section should remain undamaged. When such a specimen is placed in the fixative, the surface area of the cellular cementum exposed to the fixative is greatly increased. The fixative now has a very short distance to diffuse to reach the cells via lacunae and canaliculi which have been exposed on both cut surfaces of the section. The fixative will now reach cells located at all points between the surface of the cementum and the surface of the dentine at the same time. Many of the large irregular lacunae appeared dark both before and after mounting media infiltration and also appeared dark after embedment in methyl methacrylate. Other large irregular lacunae became infiltrated with the mounting medium or methly methacrylate and appeared translucent after infiltration. No casts corresponding in size or configuration to these lacunae were seen in the SEM preparations. These large irregular lacunae were probably infiltrated because the lacunae and canaliculi were made accessible to the mounting media or methyl methacrylate by exposure through sectioning rather than because they had a continuous interconnecting canalicular system which ultimately opened onto the surface of the cementum. Although these lacunae had associated canaliculi, the canaliculi differed from those associated with the bonelike lacunae in that they presented a tree-like branching configuration. Such a configuration in itself, however, would not mitigate against a continuous interlacunar canalicular system. The nature of the large irregular lacunae and their contents must remain speculative. Cementoblasts and epithelial rest cells (Lester, 1969; Jande and Belanger, 1970) are known to become entrapped in cellular cementum. Of these two cellular elements, only cementoblasts are known to produce cellular processes which could be surrounded by matrix to produce canaliculi. Therefore, if these lacunae contain or contained cells, it would appear most likely that the cells were large cementocytes or that more than one cementocyte resides in these lacunae. In either case, the processes of these cells, for some reason, never established contact with neighboring cells. Although it seems unlikely, the possibility that these darkened regions represent isolated modifications of the extracellular matrix rather than actual lacunae cannot be totally excluded. The observation of the sponge-like casts near the surface of the dentine was surprising because light microscopicobservations of the sections prior to matrix removal did not suggest their presence. The size and configu- CANALICULAR SYSTEM IN CELLULAR CEMENTUM ration of these casts suggest that they do not represent an aberrant form of lacunae. They are much larger than both the bone-like and the large irregular lacunae, and the reticular configuration of the casts does not correspond to the canalicular network observed in cellular cementum in the present study or the canalicular network previously observed in bone (Curtis et al., 1985). Since unmineralized areas of varying size have been previously observed near the surface of the dentine (Furseth, 19691, it is possible that these casts represent an infiltration of the matrix rather than an infiltration of spaces housing cells and their processes. Re-embedding the casts and sectioning them for transmission electron microscopy would answer this question. If extracellular matrix is seen in TEM preparations, then the casts would represent an infiltration of the matrix. If nothing is observed in the casts, this would suggest that the casts represent empty spaces which were previously occupied by cells. The presence of cellular elements would, certainly, confirm the latter suggestion. LITERATURE CITED Belanger, L.F. 1968 Resorption of cementum by cementwyte activity ("cememtolysis").CalcS. Tissue Res. 2229-236. 127 Curtis, T., S. Ashrafi, and D.F. Weber 1985 Canalicular communication in the cortices of human long bones. Anat. Rec. 212:336-344. Furseth, R. 1967 A microradiographic and electron microscopic study of the cementum of human deciduous teeth. Ada Odontol. Scand. 25:613-645. Furseth, R. 1969 The fine structure of the cellular cementum of young human teeth. Arch. Oral Biol. 14:1147-1158. Furseth, R. 1970 A microradiographic, light microscopic and electron microscopic study of the cementum from deciduous teeth of pigs. Acta Odontol. Scand. 28~811-831. Furseth, R. 1977 Further observations on the fine structure of cellular cementum from deciduous teeth of pigs. Calcif Tissue Res. 24239242. Gaynor, M.K., D.F. Weber, and J . Everingham 1984 A M e r e n t approach to immersion fmtion of human dental pulp and odontoblast processes. Arch Oral Biol. 29:243-248. Jande, S.S., and L.F. Belanger 1970 Fine structural study of rat molar cementum. Anat. Rec. 167:439-464. Lester, K.S. 1969 The incorporation of epithelial cells by cementum. J . Ultrastruct. Res. 27~63-87. Mathews, J.L. 1980 Bone structure and ultrastructure. In: Fundamental and Clinical Bone Physiology. M.R. Urist, ed. J.P. Lippincott, Philadelphia, p. 31. Weber, D.F., D.R. Eisenmann, and A.E. Zaki 1982 A grinding method for producing sections of large areas of plastic embedded tissues. J. Microsc. 127,271-276. Weber, D.F. 1983 A n improved technique for producing casts of the internal structure of hard tissues, including some observations on human dentine. Arch Oral Biol. 28r885-891.