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Histology of the periodontal ligament of rat mandibular incisor following root resection, with special reference to the zone of shear
ArcAs owl Bid. Vol. 21 pp. 235 to 244 Pergamon Press Ltd 1980. Printed in Great
Britain
HISTOLOGY OF THE PERIODONTAL LIGAMENT OF RAT MANDIBULAR INCISOR FOLLOWING ROOT RESECTION, WITH SPECIAL REFERENCE TO THE ZONE OF SHEAR B. K. B. BERKOMTZ,R. C. SHOREand P. SLOAN* Department of Anatomy (Oral Biology), The Medical School, University of Bristol BS ITD, England and * Department of Pathology, University of Newcastle-upon-Tyne,
England
Summuy-An ultrastructural study of the periodontal ligament following root resection of the rat incisor, when the basal proliferative tissues are removed, was undertaken to determine the location of its zone of shear. Fourteen days after root resection, the vacated socket was lined with connective tissue which, for at least 1 mm behind the base of the erupting tooth, was similar in structure and dimensions to the normal functional ligament above the base of the tooth. More distant from the tooth base, some loss of structure and organisation was evident, probably related to loss of function. By the 23rd day after resection, the socket began to fill with loose connective tissue and by the 29th there was bone deposition within the socket. The results may indicate that the zone of shear is located immediately adjacent to the tooth and not towards the mid-region of the ligament.
INTRODUCTION Considerable information is available concerning the ultrastructure of the periodontal ligament of the continuously growing rodent incisor, with both transmission and scanning electron microscopy. Such information deals with the detailed organization of the collagen fibres (e.g. Sloan, Shellis and Berkovitz, 1976; Sloan, 1978) and the morphology of the fibroblasts (Ten Cate, 1972; Beer&en, Everts and van den Hooff, 1974; Frank, Fellinger and Steuer, 1976; Shore and Berkovitz, 1979a). Following root resection, when the proliferative basal tissues are removed, the rat incisor continues to erupt (Bryer, 1957; Kostlti, Thoiovh and Skach, 1960; Berkovitz and Thomas, 1969; Pitaru et al., 1976) and much has been made of this observation in elucidating the mechanism of tooth eruption (reviewed by Berkovitz, 1976). Though it has been assumed that the periodontal ligament of root-resected teeth is similar to that of normal unoperated teeth, no evidence. of this is available. During eruption, the tissues of the rodent incisor are continuously formed at the base by the proliferative organ. It has been suggested that the fibroblasts of the ligament migrate occlusally from this zone at the same rate as the tooth (Beertsen, 1975), or at an even faster rate, (Zajicek, 1974) and in so doing generate ‘the eruptive force. Others have proposed that not all the cells of the ligament migrate in this way but that a zone of shear occurs towards the mid-region of the ligament, the inner dental zone moving with the tooth, the outer alveolar zone remaining behind (Melcher, 1967; Beer&n, 1973; Beertsen and Everts, 1977). It might be expected, therefore, that following root resection, the vacated socket would be lined by connective tissue representing only the outer alveolar zone. Results from light microscopy suggested that, following root resection, the vacated socket was lined 235
by a vascular connective tissue continuous in an oral direction with the periodontal tissue associated with the erupting tooth segment (Berkovitz, 1971). That this lining was similar in width to normal periodontal ligament was interpreted as indicating that little of the periodontal ligament migrated with the tooth (Berkovitz, 1975). Though observations have been made on the enamel aspect of the rodent incisor following root resection (Berkovitz and Shore, 1978), no detailed studies have been reported on the periodontal ligament proper to provide information concerning this vascular connective tissue and the site of the zone of shear. A further problem concerning any periodontal ligament tissue left when the root is resected relates to whether, and for how long, any remaining collagen fibre bundles retain their orientation and insertion into the alveolar bone as Sharpey fibres. We therefore decided to undertake an ultrastructural study of the periodontal ligament of root-resected teeth. MATERIALSAND METHODS Root resection was carried out on the right mandibular incisor of male Wistar rats, average weight 250 g, as previously described (Berkovitz and Thomas, 1969). The teeth were maintained in the unimpeded state by trimming to the gingival margin every 2 days with a rotating Carborundum disc. Fourteen specimens were examined, five at 14 days and three each at 16, 23 and 29 days following root resection. Impeded and unimpeded teeth not subjected to root resection were used as controls. Animals were injected with a lethal dose of sodium pentobarbitone (Nembutal, Abbot Laboratories, Kent), perfused with 2.5 per cent glutaraldehyde in 0.1 M phosphate buffer and subsequently processed further for either transmission (TEM) or scanning electron microscopy @EM).
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B. K. B. Berkovitz, R. C. Shore and P. Sloan RESULTS I4 and 16 days
Fig. I. Diagram of root-resected lower incisor of the rat showing the positions of the four levels excised for microscopical examination. Light and transmission electron microscopy (TEM) The lower incisors were dissected out and immersed for 2 h in 2.5 per cent glutaraldehyde in 0.1 M phosphate buffer. The jaws were then demineralized at room temperature in a solution of 5 per cent EDTA (w/v), 2.5 per cent glutaraldehyde (v/v), 6.5 per cent sucrose (w/v) in phosphate buffer, pH 7.3. After 10 weeks, when demineralization was shown by radiography to be complete, 1 mm segments of the incisor were excised with a razor blade at four levels along the length of the tooth (Text Fig. 1). The segments were post-fixed for 2 h in 1 per cent osmium tetroxide in phosphate buffer, pH 7.3, dehydrated through a graded series of ethanols and epoxy-propane and embedded in Epon 812; the shape of the tissue allowed reliable orientation. 1 pm Epon sections in both transverse and longitudinal planes and stained with Toluidine Blue were studied by light microscopy; ultrathin sections of selected areas were stained with uranyl acetate and lead citrate and examined by electron microscopy. Scanning electron microscopy (SEM) Teeth were prepared by the following methods: (a) For examination of the attachment bone. The mandibles were defleshed and the parts containing the incisor teeth separated. The labial and part of the lateral alveolar bone from the incisor socket were dissected away with a sharp scalpel and the specimens placed into a freshly made solution of 5 per cent sodium hypochlorite. After three days, the tooth or tooth fragment had separated from the alveolus leaving the socket wall intact. The pieces of alveolus were then washed in a continuous stream of warm water for 2 days, air-dried and mounted on stubs with the periodontal attachment surface uppermost. (b) For examination of the connective tissues. The jaws were demineralized using the same procedure as for TEM processing. Slices of tissue, cut in the transverse plane and approx. 2 mm thick, were then prepared with a razor blade. Each slice was hemisected by making a further cut in the longitudinal axis of the tooth. The blocks were rinsed in 0.1 M phosphate buffer, dehydrated in ethanol, critical point dried and mounted on stubs (Sloan et al., 1976). All specimens were coated with carbon and goldpalladium by evaporation and examined in a Cambridge 600 scanning electron microscope. Results were recorded photographicaily using a stereo-pair technique.
Level I. The ligament associated with the rootresected tooth appeared to be similar to controls (c.f. Plate Figs. 2 and 3). Thus, it was composed of an inner dental zone of fibroblasts orientated parallel to the long axis of the tooth, and an outer vascular zone. SEM of demineralized specimens in this region showed a normal ligament lO&150pm wide (Plate Fig. 4). TEM showed the ligament to have a normal appearance (Plate Figs. 5 and 6). Thus, the fibroblasts contained the intracellular organelles associated with protein synthesis, membrane-bound collagen profiles and microtubules; between the cells, collagen was arranged in a series of thin sheets of varying orientation. Levels 2 and 3. The connective tissue lining the vacated socket beneath the root-resected tooth was continuous with the periodontal ligament occlusally and similar to it in appearance and dimensions (Plate Fig. 7) for at least 1 mm behind the tooth (Plate Fig. 8). With SEM the residual periodontal ligament lining the vacated socket resembled the normal periodontal ligament, its structure consisting of an inner fibrous layer lining the socket space and an outer bone-related layer containing large blood vessels (Plate Fig. 10). The fibres within the layers appeared to be less highly organized than in the normal ligament. However, they were generally arranged as radially disposed sheets running in the long axis of the tooth. There was evidence of bone deposition on the surface of the socket wall. The TEM appearance was similar to level 1, though in some areas there was a decrease in collagen fibre density and a corresponding increase in ground substance. More basally, areas exhibited a lower cell density. Few, if any, collagen fibres were inserted into the alveolar walI beneath the erupting root-resected tooth. There were degenerative changes in some of the myelinated nerve fibres within the nerve bundles adjacent to the alveolar bone. A band of connective tissue formed a limiting membrane at the base of the tooth and merged at its margins with the periodontal ligament (Fig. 7). Leuel 4. The structure of the ligament appeared to change, with a slight reduction in width and in cell density (Fig. 9) and with bone deposition along the surface of the alveolar bone. The space vacated by the tooth was filled with an amorphous substance at the edges of which were found blood cells (Fig. 9). In the hypochlorite-treated specimens, no differences were observed with SEM in the appearance of surface alveolar bone between sockets associated with root-resected and control teeth. The basal two-thirds of the alveolar wall in both cases was deeply convoluted with many rounded projections (Plate Fig. 11); the crestal one-third was much flatter (Plate Fig. 12). The peaks of the projections and large areas of the crestal bone were covered with low rounded projections 7-2Opm in diameter, which we interpret as attachment sites of Sharpey fibres. The remaining areas were predominantly covered with either shallow lacunae or a branching fibrous pattern. ?3 Days By this time, the base of the erupting tooth segment approached the level of the alveolar crest.
Root-resected incisor periodontium Levels 2 and 3. The connective tissue lining the vacated socket beneath the first molar was composed of fibroblasts containing the organelles associated with protein synthesis and of well-organized sheets of collagen fibrils. Extending from the surface of this tissue into the amorphous material filling the socket, there were strands of elongated fibroblasts; many white blood cells were present between these strands. Level 4. Compared with levels 2 and 3, a more advanced stage of infilling of the vacated socket was evident beneath the third molar. Compared with normal ligament, there had been an increase in width and a loss of cell orientation (Plate Figs. 13 and 14). Blood spaces were evident as well as differentiation of a lining layer around the remaining tooth space (Fig. 13). With TEM, abundant collagen was evident between the cells but was not organized into sheets or bundles. In hypochlorite-treated root-resected specimens viewed with SEM, the bone surface at the alveolar crest was similar to that of control specimens, whereas the surface of the vacated socket was characterized by the presence of numerous mineral clusters l-3 pm in diameter (Plate Fig. 15) suggestive of rapid bone deposition (Boyde, 1972). Shallow, smoothbased circular or oval depressions S-10pm in diameter were commonly associated with these mineral clusters and were probably sites occupied by osteoblasts in vivo.
29 Days By this time, the tooth had exfoliated. At level 4, further infilling of the socket with connective tissue had occurred, the remaining spaces having a distinct cellular lining of closely-packed cuboidal cells (Plate Fig. 16) adjacent to which strands of fibrin were seen. The cells contained prominent amounts of rough endoplasmic reticulum and possessed cytoplasmic projections into the vacated socket. Little collagen was evident around these cells. Areas within the connective tissue similar to those seen in demineralized forming bone, with thin, randomly arranged, closely packed electron-dense fibrils, were observed. These areas were surrounded by cuboidal cells with the characteristics of osteoblasts, displaying a prominent Golgi complex and endoplasmic reticulum. Within the remaining socket spaces, there were many macrophages containing vesicles filled with an amorphous material similar to that seen extracellularly (Plate Fig. 17). As macrophage pseudopodia were also observed investing red blood cells, we assumed that the red cells were being phagocytosed. At level 3, the tissue showed a slightly less advanced stage of infilling with no distinct lining cells but many white blood cells. In hypochlorite-treated specimens viewed with SEM, no projections were seen at the surface of alveolar crest bone (Plate Fig. 18). DISCUSSION
Our study shows that the ultrastructure of the periodontal ligament of root-resected teeth is similar to that of normal teeth, a finding consistent with the hypothesis that the eruptive mechanism resides within the periodontal ligament.
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Three findings have led to the interpretation that a relatively well-localized zone of shear occurs towards the mid-region of the rodent incisor periodontal ligament, the inner tooth-related part moving with the tooth, the outer alveolar portion remaining behind: (1) Division of the ligament into two parts on morphological grounds (Beer&n et al., 1974; Beertsen, 1975). (2) The presence of a relatively high concentration of intracellular collagen profiles within fibroblasts situated towards the mid-region of the ligament (Beertsen and Everts, 1977). (3) Movement of both collagen and cells of the inner, tooth-related part at the same rate as tooth eruption (Beertsen, 1973, 1975; Beertsen and Everts, 1977). However, recent studies have cast some doubt as to the validity of such an interpretation (Shore and Berkovitz, 1979a, b). Assuming that eruption of the root-resected tooth is similar to that of normal teeth, an assumption partly supported by the findings that eruption rates of root-resected and control teeth are similar and can be equally affected by drugs (Berkovitz and Thomas, 1969; Berkovitz, 1972), our present findings may be relevant to the location of this zone of shear. If the zone of shear is localized towards the mid-region of the periodontal ligament, the vacated socket following root resection would be expected to be lined by a narrow zone of connective tissue representing only the original outer alveolar zone of the ligament. The vacated socket was, however, initially lined by a connective tissue layer comparable in structure and dimensions to that of the complete normal ligament; two possible interpretations can be offered to explain this finding: 1. A zone of shear may occur towards the mid-region of the ligament but the full thickness is restored, perhaps as the result of proliferation, or migration either from the perivascular connective tissue of the remaining outer alveolar zone, or from the connective tissue layer immediately beneath the base of the erupting tooth. 2. A zone of shear may occur immediately adjacent to the tooth, the majority of the ligament (with the probable exception of the cementoblast layer) being left behind as the tooth erupts. If the first explanation was correct, a constricted region of connective tissue would occur immediately behind the base of the tooth before restoration to its full thickness; this was not the case. It seems unlikely that restoration was achieved as a result of proliferation from the connective tissue layer immediately beneath the base of the erupting tooth in view of the amount of tissue that would have to be produced and the lack of any significant mitotic activity in this region. The second explanation would imply the existence of a zone of shear but would place it adjacent to the cementum and would suggest that protein turnover in the bulk of the ligament is not directly related to shear. If the periodontal tissue immediately adjacent to the cementum surface had properties different from those of the remaining ligament, there would be a basis for explaining the localization of shear in this region. Difficulties in understanding the relationship between zones of shear, tooth movements and protein turnover derive from our ignorance of both the eruptive (e.g. Berkovitz, 1976) and supportive (Picton, 1969) mechanisms. We know little about the func-
B. K. B. Berkovitz. R. C. Shore and P. Sloan
238
tional state of collagen within the ligament at any given time. As radiographic and biochemical studies indicate a relatively high and uniform turnover (e.g. Beertsen and Everts, 1977; Sodek et al., 1977; Orlowski. 1978), the question arises whether the collagen fibrils comprising a fibre (or sheet of collagen) are replaced or remodelled in such a manner as to leave the fibre continuous. Are there a few or many breaks along each fibre and do these occur randomly? Such information is required for any model concerning the behaviour of the ligament during eruption. The structural changes, namely areas of reduced collagen and cell density, and alterations in collagen fibre orientation, noted in the tissue lining the vacated socket could be related to loss of the tooth and therefore ligament function; so also could the bone deposition, the loss of fibre insertron at the alveolar bone surface and the slight reduction in thickness of the connective tissue lining (Coolidge, 1937). Bone formation was evident within the connective tissue of the tooth space 29 days after root resection. It was not determined, however, whether this bone represents outgrowths from the alveolar wall or whether isolated areas can also form within the remaining ligament. If isolated areas can form. it is not clear whether the collagenous matrix for such new bone is derived from fibroblasts or osteoblasts of the ligament. Twenty-nine days after operation the cuboidal cells lining the residual tooth space appeared to be organized into an epithelial-like layer. They had the ultrastructural characteristics of cells actively secreting protein. As their apparent high secretory activity was not associated with proportionate amounts of collagen in their immediate vicinity, it is possible that they have some role other than collagen production during the infilling of the socket.
Berkovitz B. K. B. 1975. Mechanisms of tooth eruption. In: Applied Physiology of the Mouth (Edited by Lavelle C. L. B.) PP. 99-123. John Wright. Bristol. Berkovitz B. K. B. 1976. Theories of tooth eruption. In: The Eruption and Occlusion of Teeth. (Edited by Poole D. F. G. and Stack M. V.) pp. 193-204. Butterworth, London. Berkovitz B. K. B. and Shore R. C. 1978. The ultrastructure of the enamel aspect of the rat incisor periodontium in normal and root resected teeth. Archs oral Biol. 23, 68 1689. Berkovitz B. K. B. and Thomas N. R. 1969. Unimpeded eruption in the root resected lower incisor of the rat with a preliminary note on root transection. Archs oral Biol. 14, 771-780. Boyde A. 1972. Scanning electron microscopic studies of bone. In: The Biochemistry and Physiology of Bone I. (Edited by Bourne G. H.) pp. 259-309. Academic Press, London. Bryer L. W. 1957. An experimental evaluation of the physiology of tooth eruption. Int. dent. J. 7,432478. Coolidge E. D. 1937. The thickness of the human periodontal membrane. J. Am. dent.’ Ass. 24, 126&1270. Frank R. M., Fellinger E. et Steuer P. 1976. Ultrastructure du ligament alvtolo-dentaire du rat. J. Biol. buccale 4, .L
295-313.
Kostlan J., Thor&a J. and Skach M. 1960. Erupce Uodaveho jubo po resekci jeho rdstove jdny. Cs &omar. 6, 401410. Melcher A. H. 1967. Remodelling of the periodontal ligament during eruption of the rat incisor. Archs oral Biol. 12, 164991651. Orlowski W. A. 1978. Biochemical study of collagen turnover in rat incisor periodontal ligament. Archs oral Biol. 23, 1163-l 165. Picton D. C. A. 1969. The effect of external forces on the periodontium. In: The Biology of the Periodontium. (Edited by Melcher A. H. and Bowen W. H) pp. 363421. Academic Press, London, Pitaru S., Michaeli Y., Zajicek G. and Weinreb M. M. 1976. Role of attrition and occlusal contact in the physiology of the rat incisor. IX. The part played by the periodontal ligament in the eruptive processes. J. dent. Res. 55, 8 19-824.
Acknowledgements-We are grateful to Research Council for funding this project.
the
Medical
REFERENCES
Beertsen W. 1973. Tissue dynamics in the periodontal ligament of the mandibular incisor of the mouse: A preliminary report. Archs oral Biol. 18, 61-66. Beertsen W. 1975. Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Archs oral Biol. 20, 659-666. Beertsen W. and Everts V. 1977. The site of remodelling of collagen in the periodontal ligament of the mouse incisor. Anut. Rec. 189, 479498. Beertsen W., Everts V. and van den Ho08 A. 1974. Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. Archs oral Biol. 19, 1087-1098. Berkovitz B. K. B. 1971. The healing process in the incisor tooth socket of the rat following root resection and exfoliation. Archs oral Biol. 16, 1045-1054. Berkovitz B. K. B. 1972. The effect of demecolcine and of triethanomelamine on the unimpeded eruption rate of normal and root resected incisor teeth in rats. Archs oral Biol. 17, 937-947.
Shore R. C. and Berkovitz B. K. B. 1979a. An ultrastructural study of periodontal ligament fibroblasts in relation to their possible role in-tooth eruption and intracellular collagen degradation in the rat. Archs oral Biol. 24, 1555164. Shore R. C. and Berkovitz B. K. B. 1979b. Model to explain apparent occlusal movement of extracellular protein of periodontal ligament of rat incisor. Archs oral Biol. 24, 861-862.
Sloan P. 1978. Scanning electron microscopy of the collagen fibre architecture of the rabbit incisor periodontium. Archs oral Biol. 23, 567-572.
Sloan P., Shellis R. P. and Berkovitz B. K. B. 1976. Effect of specimen preparation on the appearance of the rat periodontal ligament in the scanning electron microscope. Archs oral Biol. 21, 633635. Sodek J. Brunette D. M., Feng J., Heersche J. N. M., Limeback H. F., Melcher A. H. and Ng B. 1977. Collagen synthesis is a major component of protein synthesis in the periodontal ligament in various species. Archs oral Bid/. 22, 647653. Ten Cate A. R. 1972. Morphological studies of fibrocytes in connective tissue undergoing rapid remodelling. J. Anat. 112,401414.
Zajicek G. 1974. Fibroblast cell kinetics in the periodontal ligament of the mouse. Cell Tissue Kinet. 1, 479492.
Root-resected incisor periodontium
Plates l-4 overleaf.
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Plate 1. Fig. 2. Light micrograph of longitudinal section of control periodontal ligament. Tooth (T). Alveolar bone (A). Inner dental zone of periodontal ligament (I). Outer alveolar zone of ligament (0). There is a vascular space within this zone. x 280 Fig. 3. Light micrograph of longitudinal section of periodontal ligament of a root-resected tooth 14 days after operation. Tooth (T). Alveolar bone (A). Inner dental zone of ligament (I). Outer alveolar zone of ligament (0). x 340 Fig. 4. SEM of demineralized transverse section of periodontal ligament of root-resected tooth at level 1, 14 days after operation. The ligament appears normal. Alveolar bone (A), ligament (L), tooth (T) and pulp space (P). x 130 Fig. 5. TEM of longitudinal section of inner dental zone of ligament of a root-resected tooth, 14 days after operation. Note the elongated fibroblasts containing all the intracytoplasmic organelles associated with protein synthesis. Between the cells are collagen fibrils cut both longitudinally and transversely. X 6800 Fig. 6. TEM of longitudinal section of inner dental zone of a control ligament at level I. Note the arrangement and appearance of fibroblasts and collagen fibrils similar to those in Fig. 5. x 5100 Plate 2. Fig. 7. Light micrograph of longitudinal section of periodontal ligament associated with the base of a root-resected tooth (T). Note the overall similarity in width and organization of the ligament associated with the tooth and with the vacated socket (S). Connective tissue (C) lies beneath the base of the tooth. Alveolar bone (A). x 3 IO Fig. 8. Light micrograph of longitudinal section of periodontal ligament associated with the vacated socket 1mm below the base of a root-resected tooth (i.e. level 3), 14 days after operation. Note blood cells in the vacated socket (S). Alveolar bone (A). x 280 Fig. 9. Light micrograph of longitudinal section of periodontal ligament associated with the vacated socket of a root-resected tooth at level 4, 14 days after operation. Bone is being deposited on alveolar wall. Vacated socket (S). Alveolar bone (A). x 220 Fig. 10. SEM of deminerahsed transverse section of periodontal ligament at level 2 just beneath the base of the root resected tooth, 14 days after operation. Inner fibrous (I) and outer vascular layers (0) are evident. At the surface of the alveolus (A) a layer of recently formed bone has been deposited (B). Tooth space (S). x 330 Plate 3. Fig. 11. SEM of the alveolar wall of a vacated socket at level 4, 14 days after operation, following treatment with sodium hypochlorite. Note the deep channels which originally contained blood vessels. X 130 Fig. 12. SEM of crestal region of the alveolar wall 14 days after operation, following treatment with hypochlorite. The wall is relatively smooth and is covered with rounded elevations interpreted as mineralized parts of Sharpey fibres. x 660 Fig. 13. Light micrograph of transverse section of the periodontal ligament associated with the vacated socket at level 4, 23 days after operation. Note the lining layer around the tooth space (S), and appearance of blood spaces within the connective tissue which is twice the width of the normal ligament. Alveolar bone lies just beyond the field of view at the lower margin. x 110 Fig. 14. Light micrograph of transverse section of control periodontal ligament at level 4. Alveolar bone (A). Tooth (T). x 210 Plate 4. Fig. 15. SEM of alveolar surface of a vacated socket 23 days after operation, following treatment with hypochlorite. The surface is covered with mineral clusters. x 1300 Fig. 16. TEM of cell lining of a vacated socket at level 4, 29 days after operation. Note the cuboidal shape and the fine cytoplasmic processes (arrowed) projecting into the tooth space (S). There is very little collagen around the cell. x 1800 Fig. 17. TEM of macrophage within a vacated socket 29 days after operation. There are numerous large vacuoles within the cytoplasm containing material similar to that seen extracellularly. Part of a red blood corpuscle (arrow) is adjacent to the cell surface. x 4100 Fig. 18. SEM of alveolar surface in crestal region of a vacated socket 29 days after operation following treatment with hypochiorite. The ends of Sharpey fibres are obscured by amorphous material (compare Fig. 12). x 1300
Root-resected
incisor periodontium
Plate 1.
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B. K. B. Berkovitz, R. C. Shore and P. Sloan
Plate 2.
Root-resected
incisor periodontium
Plate 3.
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Britain
HISTOLOGY OF THE PERIODONTAL LIGAMENT OF RAT MANDIBULAR INCISOR FOLLOWING ROOT RESECTION, WITH SPECIAL REFERENCE TO THE ZONE OF SHEAR B. K. B. BERKOMTZ,R. C. SHOREand P. SLOAN* Department of Anatomy (Oral Biology), The Medical School, University of Bristol BS ITD, England and * Department of Pathology, University of Newcastle-upon-Tyne,
England
Summuy-An ultrastructural study of the periodontal ligament following root resection of the rat incisor, when the basal proliferative tissues are removed, was undertaken to determine the location of its zone of shear. Fourteen days after root resection, the vacated socket was lined with connective tissue which, for at least 1 mm behind the base of the erupting tooth, was similar in structure and dimensions to the normal functional ligament above the base of the tooth. More distant from the tooth base, some loss of structure and organisation was evident, probably related to loss of function. By the 23rd day after resection, the socket began to fill with loose connective tissue and by the 29th there was bone deposition within the socket. The results may indicate that the zone of shear is located immediately adjacent to the tooth and not towards the mid-region of the ligament.
INTRODUCTION Considerable information is available concerning the ultrastructure of the periodontal ligament of the continuously growing rodent incisor, with both transmission and scanning electron microscopy. Such information deals with the detailed organization of the collagen fibres (e.g. Sloan, Shellis and Berkovitz, 1976; Sloan, 1978) and the morphology of the fibroblasts (Ten Cate, 1972; Beer&en, Everts and van den Hooff, 1974; Frank, Fellinger and Steuer, 1976; Shore and Berkovitz, 1979a). Following root resection, when the proliferative basal tissues are removed, the rat incisor continues to erupt (Bryer, 1957; Kostlti, Thoiovh and Skach, 1960; Berkovitz and Thomas, 1969; Pitaru et al., 1976) and much has been made of this observation in elucidating the mechanism of tooth eruption (reviewed by Berkovitz, 1976). Though it has been assumed that the periodontal ligament of root-resected teeth is similar to that of normal unoperated teeth, no evidence. of this is available. During eruption, the tissues of the rodent incisor are continuously formed at the base by the proliferative organ. It has been suggested that the fibroblasts of the ligament migrate occlusally from this zone at the same rate as the tooth (Beertsen, 1975), or at an even faster rate, (Zajicek, 1974) and in so doing generate ‘the eruptive force. Others have proposed that not all the cells of the ligament migrate in this way but that a zone of shear occurs towards the mid-region of the ligament, the inner dental zone moving with the tooth, the outer alveolar zone remaining behind (Melcher, 1967; Beer&n, 1973; Beertsen and Everts, 1977). It might be expected, therefore, that following root resection, the vacated socket would be lined by connective tissue representing only the outer alveolar zone. Results from light microscopy suggested that, following root resection, the vacated socket was lined 235
by a vascular connective tissue continuous in an oral direction with the periodontal tissue associated with the erupting tooth segment (Berkovitz, 1971). That this lining was similar in width to normal periodontal ligament was interpreted as indicating that little of the periodontal ligament migrated with the tooth (Berkovitz, 1975). Though observations have been made on the enamel aspect of the rodent incisor following root resection (Berkovitz and Shore, 1978), no detailed studies have been reported on the periodontal ligament proper to provide information concerning this vascular connective tissue and the site of the zone of shear. A further problem concerning any periodontal ligament tissue left when the root is resected relates to whether, and for how long, any remaining collagen fibre bundles retain their orientation and insertion into the alveolar bone as Sharpey fibres. We therefore decided to undertake an ultrastructural study of the periodontal ligament of root-resected teeth. MATERIALSAND METHODS Root resection was carried out on the right mandibular incisor of male Wistar rats, average weight 250 g, as previously described (Berkovitz and Thomas, 1969). The teeth were maintained in the unimpeded state by trimming to the gingival margin every 2 days with a rotating Carborundum disc. Fourteen specimens were examined, five at 14 days and three each at 16, 23 and 29 days following root resection. Impeded and unimpeded teeth not subjected to root resection were used as controls. Animals were injected with a lethal dose of sodium pentobarbitone (Nembutal, Abbot Laboratories, Kent), perfused with 2.5 per cent glutaraldehyde in 0.1 M phosphate buffer and subsequently processed further for either transmission (TEM) or scanning electron microscopy @EM).
236
B. K. B. Berkovitz, R. C. Shore and P. Sloan RESULTS I4 and 16 days
Fig. I. Diagram of root-resected lower incisor of the rat showing the positions of the four levels excised for microscopical examination. Light and transmission electron microscopy (TEM) The lower incisors were dissected out and immersed for 2 h in 2.5 per cent glutaraldehyde in 0.1 M phosphate buffer. The jaws were then demineralized at room temperature in a solution of 5 per cent EDTA (w/v), 2.5 per cent glutaraldehyde (v/v), 6.5 per cent sucrose (w/v) in phosphate buffer, pH 7.3. After 10 weeks, when demineralization was shown by radiography to be complete, 1 mm segments of the incisor were excised with a razor blade at four levels along the length of the tooth (Text Fig. 1). The segments were post-fixed for 2 h in 1 per cent osmium tetroxide in phosphate buffer, pH 7.3, dehydrated through a graded series of ethanols and epoxy-propane and embedded in Epon 812; the shape of the tissue allowed reliable orientation. 1 pm Epon sections in both transverse and longitudinal planes and stained with Toluidine Blue were studied by light microscopy; ultrathin sections of selected areas were stained with uranyl acetate and lead citrate and examined by electron microscopy. Scanning electron microscopy (SEM) Teeth were prepared by the following methods: (a) For examination of the attachment bone. The mandibles were defleshed and the parts containing the incisor teeth separated. The labial and part of the lateral alveolar bone from the incisor socket were dissected away with a sharp scalpel and the specimens placed into a freshly made solution of 5 per cent sodium hypochlorite. After three days, the tooth or tooth fragment had separated from the alveolus leaving the socket wall intact. The pieces of alveolus were then washed in a continuous stream of warm water for 2 days, air-dried and mounted on stubs with the periodontal attachment surface uppermost. (b) For examination of the connective tissues. The jaws were demineralized using the same procedure as for TEM processing. Slices of tissue, cut in the transverse plane and approx. 2 mm thick, were then prepared with a razor blade. Each slice was hemisected by making a further cut in the longitudinal axis of the tooth. The blocks were rinsed in 0.1 M phosphate buffer, dehydrated in ethanol, critical point dried and mounted on stubs (Sloan et al., 1976). All specimens were coated with carbon and goldpalladium by evaporation and examined in a Cambridge 600 scanning electron microscope. Results were recorded photographicaily using a stereo-pair technique.
Level I. The ligament associated with the rootresected tooth appeared to be similar to controls (c.f. Plate Figs. 2 and 3). Thus, it was composed of an inner dental zone of fibroblasts orientated parallel to the long axis of the tooth, and an outer vascular zone. SEM of demineralized specimens in this region showed a normal ligament lO&150pm wide (Plate Fig. 4). TEM showed the ligament to have a normal appearance (Plate Figs. 5 and 6). Thus, the fibroblasts contained the intracellular organelles associated with protein synthesis, membrane-bound collagen profiles and microtubules; between the cells, collagen was arranged in a series of thin sheets of varying orientation. Levels 2 and 3. The connective tissue lining the vacated socket beneath the root-resected tooth was continuous with the periodontal ligament occlusally and similar to it in appearance and dimensions (Plate Fig. 7) for at least 1 mm behind the tooth (Plate Fig. 8). With SEM the residual periodontal ligament lining the vacated socket resembled the normal periodontal ligament, its structure consisting of an inner fibrous layer lining the socket space and an outer bone-related layer containing large blood vessels (Plate Fig. 10). The fibres within the layers appeared to be less highly organized than in the normal ligament. However, they were generally arranged as radially disposed sheets running in the long axis of the tooth. There was evidence of bone deposition on the surface of the socket wall. The TEM appearance was similar to level 1, though in some areas there was a decrease in collagen fibre density and a corresponding increase in ground substance. More basally, areas exhibited a lower cell density. Few, if any, collagen fibres were inserted into the alveolar walI beneath the erupting root-resected tooth. There were degenerative changes in some of the myelinated nerve fibres within the nerve bundles adjacent to the alveolar bone. A band of connective tissue formed a limiting membrane at the base of the tooth and merged at its margins with the periodontal ligament (Fig. 7). Leuel 4. The structure of the ligament appeared to change, with a slight reduction in width and in cell density (Fig. 9) and with bone deposition along the surface of the alveolar bone. The space vacated by the tooth was filled with an amorphous substance at the edges of which were found blood cells (Fig. 9). In the hypochlorite-treated specimens, no differences were observed with SEM in the appearance of surface alveolar bone between sockets associated with root-resected and control teeth. The basal two-thirds of the alveolar wall in both cases was deeply convoluted with many rounded projections (Plate Fig. 11); the crestal one-third was much flatter (Plate Fig. 12). The peaks of the projections and large areas of the crestal bone were covered with low rounded projections 7-2Opm in diameter, which we interpret as attachment sites of Sharpey fibres. The remaining areas were predominantly covered with either shallow lacunae or a branching fibrous pattern. ?3 Days By this time, the base of the erupting tooth segment approached the level of the alveolar crest.
Root-resected incisor periodontium Levels 2 and 3. The connective tissue lining the vacated socket beneath the first molar was composed of fibroblasts containing the organelles associated with protein synthesis and of well-organized sheets of collagen fibrils. Extending from the surface of this tissue into the amorphous material filling the socket, there were strands of elongated fibroblasts; many white blood cells were present between these strands. Level 4. Compared with levels 2 and 3, a more advanced stage of infilling of the vacated socket was evident beneath the third molar. Compared with normal ligament, there had been an increase in width and a loss of cell orientation (Plate Figs. 13 and 14). Blood spaces were evident as well as differentiation of a lining layer around the remaining tooth space (Fig. 13). With TEM, abundant collagen was evident between the cells but was not organized into sheets or bundles. In hypochlorite-treated root-resected specimens viewed with SEM, the bone surface at the alveolar crest was similar to that of control specimens, whereas the surface of the vacated socket was characterized by the presence of numerous mineral clusters l-3 pm in diameter (Plate Fig. 15) suggestive of rapid bone deposition (Boyde, 1972). Shallow, smoothbased circular or oval depressions S-10pm in diameter were commonly associated with these mineral clusters and were probably sites occupied by osteoblasts in vivo.
29 Days By this time, the tooth had exfoliated. At level 4, further infilling of the socket with connective tissue had occurred, the remaining spaces having a distinct cellular lining of closely-packed cuboidal cells (Plate Fig. 16) adjacent to which strands of fibrin were seen. The cells contained prominent amounts of rough endoplasmic reticulum and possessed cytoplasmic projections into the vacated socket. Little collagen was evident around these cells. Areas within the connective tissue similar to those seen in demineralized forming bone, with thin, randomly arranged, closely packed electron-dense fibrils, were observed. These areas were surrounded by cuboidal cells with the characteristics of osteoblasts, displaying a prominent Golgi complex and endoplasmic reticulum. Within the remaining socket spaces, there were many macrophages containing vesicles filled with an amorphous material similar to that seen extracellularly (Plate Fig. 17). As macrophage pseudopodia were also observed investing red blood cells, we assumed that the red cells were being phagocytosed. At level 3, the tissue showed a slightly less advanced stage of infilling with no distinct lining cells but many white blood cells. In hypochlorite-treated specimens viewed with SEM, no projections were seen at the surface of alveolar crest bone (Plate Fig. 18). DISCUSSION
Our study shows that the ultrastructure of the periodontal ligament of root-resected teeth is similar to that of normal teeth, a finding consistent with the hypothesis that the eruptive mechanism resides within the periodontal ligament.
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Three findings have led to the interpretation that a relatively well-localized zone of shear occurs towards the mid-region of the rodent incisor periodontal ligament, the inner tooth-related part moving with the tooth, the outer alveolar portion remaining behind: (1) Division of the ligament into two parts on morphological grounds (Beer&n et al., 1974; Beertsen, 1975). (2) The presence of a relatively high concentration of intracellular collagen profiles within fibroblasts situated towards the mid-region of the ligament (Beertsen and Everts, 1977). (3) Movement of both collagen and cells of the inner, tooth-related part at the same rate as tooth eruption (Beertsen, 1973, 1975; Beertsen and Everts, 1977). However, recent studies have cast some doubt as to the validity of such an interpretation (Shore and Berkovitz, 1979a, b). Assuming that eruption of the root-resected tooth is similar to that of normal teeth, an assumption partly supported by the findings that eruption rates of root-resected and control teeth are similar and can be equally affected by drugs (Berkovitz and Thomas, 1969; Berkovitz, 1972), our present findings may be relevant to the location of this zone of shear. If the zone of shear is localized towards the mid-region of the periodontal ligament, the vacated socket following root resection would be expected to be lined by a narrow zone of connective tissue representing only the original outer alveolar zone of the ligament. The vacated socket was, however, initially lined by a connective tissue layer comparable in structure and dimensions to that of the complete normal ligament; two possible interpretations can be offered to explain this finding: 1. A zone of shear may occur towards the mid-region of the ligament but the full thickness is restored, perhaps as the result of proliferation, or migration either from the perivascular connective tissue of the remaining outer alveolar zone, or from the connective tissue layer immediately beneath the base of the erupting tooth. 2. A zone of shear may occur immediately adjacent to the tooth, the majority of the ligament (with the probable exception of the cementoblast layer) being left behind as the tooth erupts. If the first explanation was correct, a constricted region of connective tissue would occur immediately behind the base of the tooth before restoration to its full thickness; this was not the case. It seems unlikely that restoration was achieved as a result of proliferation from the connective tissue layer immediately beneath the base of the erupting tooth in view of the amount of tissue that would have to be produced and the lack of any significant mitotic activity in this region. The second explanation would imply the existence of a zone of shear but would place it adjacent to the cementum and would suggest that protein turnover in the bulk of the ligament is not directly related to shear. If the periodontal tissue immediately adjacent to the cementum surface had properties different from those of the remaining ligament, there would be a basis for explaining the localization of shear in this region. Difficulties in understanding the relationship between zones of shear, tooth movements and protein turnover derive from our ignorance of both the eruptive (e.g. Berkovitz, 1976) and supportive (Picton, 1969) mechanisms. We know little about the func-
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tional state of collagen within the ligament at any given time. As radiographic and biochemical studies indicate a relatively high and uniform turnover (e.g. Beertsen and Everts, 1977; Sodek et al., 1977; Orlowski. 1978), the question arises whether the collagen fibrils comprising a fibre (or sheet of collagen) are replaced or remodelled in such a manner as to leave the fibre continuous. Are there a few or many breaks along each fibre and do these occur randomly? Such information is required for any model concerning the behaviour of the ligament during eruption. The structural changes, namely areas of reduced collagen and cell density, and alterations in collagen fibre orientation, noted in the tissue lining the vacated socket could be related to loss of the tooth and therefore ligament function; so also could the bone deposition, the loss of fibre insertron at the alveolar bone surface and the slight reduction in thickness of the connective tissue lining (Coolidge, 1937). Bone formation was evident within the connective tissue of the tooth space 29 days after root resection. It was not determined, however, whether this bone represents outgrowths from the alveolar wall or whether isolated areas can also form within the remaining ligament. If isolated areas can form. it is not clear whether the collagenous matrix for such new bone is derived from fibroblasts or osteoblasts of the ligament. Twenty-nine days after operation the cuboidal cells lining the residual tooth space appeared to be organized into an epithelial-like layer. They had the ultrastructural characteristics of cells actively secreting protein. As their apparent high secretory activity was not associated with proportionate amounts of collagen in their immediate vicinity, it is possible that they have some role other than collagen production during the infilling of the socket.
Berkovitz B. K. B. 1975. Mechanisms of tooth eruption. In: Applied Physiology of the Mouth (Edited by Lavelle C. L. B.) PP. 99-123. John Wright. Bristol. Berkovitz B. K. B. 1976. Theories of tooth eruption. In: The Eruption and Occlusion of Teeth. (Edited by Poole D. F. G. and Stack M. V.) pp. 193-204. Butterworth, London. Berkovitz B. K. B. and Shore R. C. 1978. The ultrastructure of the enamel aspect of the rat incisor periodontium in normal and root resected teeth. Archs oral Biol. 23, 68 1689. Berkovitz B. K. B. and Thomas N. R. 1969. Unimpeded eruption in the root resected lower incisor of the rat with a preliminary note on root transection. Archs oral Biol. 14, 771-780. Boyde A. 1972. Scanning electron microscopic studies of bone. In: The Biochemistry and Physiology of Bone I. (Edited by Bourne G. H.) pp. 259-309. Academic Press, London. Bryer L. W. 1957. An experimental evaluation of the physiology of tooth eruption. Int. dent. J. 7,432478. Coolidge E. D. 1937. The thickness of the human periodontal membrane. J. Am. dent.’ Ass. 24, 126&1270. Frank R. M., Fellinger E. et Steuer P. 1976. Ultrastructure du ligament alvtolo-dentaire du rat. J. Biol. buccale 4, .L
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Kostlan J., Thor&a J. and Skach M. 1960. Erupce Uodaveho jubo po resekci jeho rdstove jdny. Cs &omar. 6, 401410. Melcher A. H. 1967. Remodelling of the periodontal ligament during eruption of the rat incisor. Archs oral Biol. 12, 164991651. Orlowski W. A. 1978. Biochemical study of collagen turnover in rat incisor periodontal ligament. Archs oral Biol. 23, 1163-l 165. Picton D. C. A. 1969. The effect of external forces on the periodontium. In: The Biology of the Periodontium. (Edited by Melcher A. H. and Bowen W. H) pp. 363421. Academic Press, London, Pitaru S., Michaeli Y., Zajicek G. and Weinreb M. M. 1976. Role of attrition and occlusal contact in the physiology of the rat incisor. IX. The part played by the periodontal ligament in the eruptive processes. J. dent. Res. 55, 8 19-824.
Acknowledgements-We are grateful to Research Council for funding this project.
the
Medical
REFERENCES
Beertsen W. 1973. Tissue dynamics in the periodontal ligament of the mandibular incisor of the mouse: A preliminary report. Archs oral Biol. 18, 61-66. Beertsen W. 1975. Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Archs oral Biol. 20, 659-666. Beertsen W. and Everts V. 1977. The site of remodelling of collagen in the periodontal ligament of the mouse incisor. Anut. Rec. 189, 479498. Beertsen W., Everts V. and van den Ho08 A. 1974. Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. Archs oral Biol. 19, 1087-1098. Berkovitz B. K. B. 1971. The healing process in the incisor tooth socket of the rat following root resection and exfoliation. Archs oral Biol. 16, 1045-1054. Berkovitz B. K. B. 1972. The effect of demecolcine and of triethanomelamine on the unimpeded eruption rate of normal and root resected incisor teeth in rats. Archs oral Biol. 17, 937-947.
Shore R. C. and Berkovitz B. K. B. 1979a. An ultrastructural study of periodontal ligament fibroblasts in relation to their possible role in-tooth eruption and intracellular collagen degradation in the rat. Archs oral Biol. 24, 1555164. Shore R. C. and Berkovitz B. K. B. 1979b. Model to explain apparent occlusal movement of extracellular protein of periodontal ligament of rat incisor. Archs oral Biol. 24, 861-862.
Sloan P. 1978. Scanning electron microscopy of the collagen fibre architecture of the rabbit incisor periodontium. Archs oral Biol. 23, 567-572.
Sloan P., Shellis R. P. and Berkovitz B. K. B. 1976. Effect of specimen preparation on the appearance of the rat periodontal ligament in the scanning electron microscope. Archs oral Biol. 21, 633635. Sodek J. Brunette D. M., Feng J., Heersche J. N. M., Limeback H. F., Melcher A. H. and Ng B. 1977. Collagen synthesis is a major component of protein synthesis in the periodontal ligament in various species. Archs oral Bid/. 22, 647653. Ten Cate A. R. 1972. Morphological studies of fibrocytes in connective tissue undergoing rapid remodelling. J. Anat. 112,401414.
Zajicek G. 1974. Fibroblast cell kinetics in the periodontal ligament of the mouse. Cell Tissue Kinet. 1, 479492.
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Plate 1. Fig. 2. Light micrograph of longitudinal section of control periodontal ligament. Tooth (T). Alveolar bone (A). Inner dental zone of periodontal ligament (I). Outer alveolar zone of ligament (0). There is a vascular space within this zone. x 280 Fig. 3. Light micrograph of longitudinal section of periodontal ligament of a root-resected tooth 14 days after operation. Tooth (T). Alveolar bone (A). Inner dental zone of ligament (I). Outer alveolar zone of ligament (0). x 340 Fig. 4. SEM of demineralized transverse section of periodontal ligament of root-resected tooth at level 1, 14 days after operation. The ligament appears normal. Alveolar bone (A), ligament (L), tooth (T) and pulp space (P). x 130 Fig. 5. TEM of longitudinal section of inner dental zone of ligament of a root-resected tooth, 14 days after operation. Note the elongated fibroblasts containing all the intracytoplasmic organelles associated with protein synthesis. Between the cells are collagen fibrils cut both longitudinally and transversely. X 6800 Fig. 6. TEM of longitudinal section of inner dental zone of a control ligament at level I. Note the arrangement and appearance of fibroblasts and collagen fibrils similar to those in Fig. 5. x 5100 Plate 2. Fig. 7. Light micrograph of longitudinal section of periodontal ligament associated with the base of a root-resected tooth (T). Note the overall similarity in width and organization of the ligament associated with the tooth and with the vacated socket (S). Connective tissue (C) lies beneath the base of the tooth. Alveolar bone (A). x 3 IO Fig. 8. Light micrograph of longitudinal section of periodontal ligament associated with the vacated socket 1mm below the base of a root-resected tooth (i.e. level 3), 14 days after operation. Note blood cells in the vacated socket (S). Alveolar bone (A). x 280 Fig. 9. Light micrograph of longitudinal section of periodontal ligament associated with the vacated socket of a root-resected tooth at level 4, 14 days after operation. Bone is being deposited on alveolar wall. Vacated socket (S). Alveolar bone (A). x 220 Fig. 10. SEM of deminerahsed transverse section of periodontal ligament at level 2 just beneath the base of the root resected tooth, 14 days after operation. Inner fibrous (I) and outer vascular layers (0) are evident. At the surface of the alveolus (A) a layer of recently formed bone has been deposited (B). Tooth space (S). x 330 Plate 3. Fig. 11. SEM of the alveolar wall of a vacated socket at level 4, 14 days after operation, following treatment with sodium hypochlorite. Note the deep channels which originally contained blood vessels. X 130 Fig. 12. SEM of crestal region of the alveolar wall 14 days after operation, following treatment with hypochlorite. The wall is relatively smooth and is covered with rounded elevations interpreted as mineralized parts of Sharpey fibres. x 660 Fig. 13. Light micrograph of transverse section of the periodontal ligament associated with the vacated socket at level 4, 23 days after operation. Note the lining layer around the tooth space (S), and appearance of blood spaces within the connective tissue which is twice the width of the normal ligament. Alveolar bone lies just beyond the field of view at the lower margin. x 110 Fig. 14. Light micrograph of transverse section of control periodontal ligament at level 4. Alveolar bone (A). Tooth (T). x 210 Plate 4. Fig. 15. SEM of alveolar surface of a vacated socket 23 days after operation, following treatment with hypochlorite. The surface is covered with mineral clusters. x 1300 Fig. 16. TEM of cell lining of a vacated socket at level 4, 29 days after operation. Note the cuboidal shape and the fine cytoplasmic processes (arrowed) projecting into the tooth space (S). There is very little collagen around the cell. x 1800 Fig. 17. TEM of macrophage within a vacated socket 29 days after operation. There are numerous large vacuoles within the cytoplasm containing material similar to that seen extracellularly. Part of a red blood corpuscle (arrow) is adjacent to the cell surface. x 4100 Fig. 18. SEM of alveolar surface in crestal region of a vacated socket 29 days after operation following treatment with hypochiorite. The ends of Sharpey fibres are obscured by amorphous material (compare Fig. 12). x 1300
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