890
The "Plaque-Free Zone" in Health and Disease: A Scanning Electron
Microscope Study Marlize T. Friedman, * Pauline M. Barber, * Nicola J. Mordan, * and Hubert N.
Newman*f
A "plaque-free" zone has been described on the enamel surface of healthy extracted teeth. This study examined this zone on chronic adult periodontitis-affected teeth (CAPT). Ten healthy controls and 16 CAPT were collected immediately after extraction, fixed, dehydrated, critical point dried, coated, and viewed by scanning electron microscopy (SEM). The "plaque-free" zone was observed in both groups as an area with few bacteria between the apical plaque border and the coronal limit of an epithelial layer on the root surface, extending to the residual periodontal ligament. On the healthy specimens, the apical plaque border consisted mainly of cocci and short rods, while on the CAPT specimens spirochetes predominated. Isolated or small groups of microorganisms were always present in the "plaque-free" zone and at its apical limit, close to or in contact with junctional epithelial cells. This zone is therefore not completely free of plaque, as suggested. It was concluded that a tissue complex, analogous to that in health on enamel, persists on the root surfaces of CAPT throughout the disease process. It comprises a discrete plaque border, a dental cuticle with sparse organisms, and an epithelium analogous to junctional epithelium. Its main function would appear to be to prevent bulk access of plaque to the surrounding tissues, including direct contact of bacteria with underlying ligament. / Periodontology 1992; 63:890-896.
Key Words: Dental plaque/epidemiology; dental plaque/microbiology; periodontitis/mi-
crobiology.
A non-staining zone is a common finding on the smooth surfaces of both healthy and diseased, stained, extracted teeth. It was first described by Bass.1 On the enamel of healthy teeth, this zone is delimited coronally by the gingival margin plaque and apically by the junctional epithelium. It corresponds, corono-apically, to the healthy crevice before extraction, and microorganisms are sparse in this
region.2 In
1973, Brady3 used scanning and transmission electron
microscopy to examine this zone on both healthy and periodontitis-affected teeth. He, too, described it as an area virtually free from microorganisms, between the apical plaque border and the attachment epithelium, and called it the "plaque-free zone" (PFZ). Although there is agreement regarding the principal structural components of this zone on enamel, there is little knowledge concerning its features on diseased teeth. While it is generally accepted that on the latter the coronal limit is the established subgingival plaque 'Electron
Microscopy Unit,
Institute of Dental
London, London UK.
Department of Periodontology.
Surgery, University
of
border4 and that the underlying cementum is covered by a dental cuticle,5'6 most views differ from Brady's description of its apical limit. On periodontitis-affected teeth, the PFZ apical limit has been described as the coronal border of the residual periodontal ligament fibers.712 Recently, however, another study13 has also suggested that, in vivo, the zone is apically limited by epithelial cells. Although this zone is now always referred to as the plaquefree zone and this term is currently accepted, its apical limit on the root surface of diseased teeth is not yet clear and the designation of plaque-free requires investigation. The aims of the present study, therefore, were to identify the components of this specific zone of the root surface of chronic adult periodontitis-affected teeth (CAPT). MATERIALS AND METHODS The material for this study comprised 10 healthy and 16 chronic adult periodontitis-affected human teeth (CAPT), collected immediately after extraction from patients referred to the Department of Maxillo-Facial Surgery and Oral Medicine. Consent was obtained from all patients, or their guardians in the case of children, concerning the study of
Volume 63 Number 11
their extracted teeth. Healthy teeth, extracted for orthodontic reasons, were obtained from patients aged 12 to 14 years, and the diseased teeth from patients aged 35 to 65 years. The inclusion criteria for CAPT were: no history of periodontal treatment for at least 1 year prior to extraction, bleeding on gentle probing, pocket depth >4 mm in relation to at least one of the approximal surfaces, and radiographie evidence of bone loss. Clinical examination was always performed by the same operator (MTF). Pocket depth was measured to the nearest millimeter with a Borodontic probe* and a Williams tip (diameter 0.5 mm), calibrated at 0.25N. All teeth were extracted without approximal instrumentation and immediately rinsed in 0.1M sodium cacodylate buffer solution (pH 7.4) to remove blood and loose debris. They were then fixed in 3% glutaraldehyde in 0.1M sodium cacodylate buffer, previously cooled at 4°C, for 3 hours. After primary fixation, teeth were post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 hours at 4°C. They were dehydrated in a graded aqueous acetone series, critical-point dried, sputter-coated with gold-palladium and viewed in a scanning electron microscope*5 or a field emission scanning electron microscope.11 The distances from the apical plaque border to the most coronal attachment epithelial cells and from the plaque border to the residual periodontal ligament fibers were measured on scanning electron micrographs.
FRIEDMAN, BARBER, MORDAN, NEWMAN
1. (Left) A healthy extracted tooth that has been stained with osmium tetroxide. Note the weakly stained band within the unstained zone
Figure
(arrows), (gm gingival margin plaque; ca contact area). Figure 2. (Right) A periodontally-affected extracted tooth that =
RESULTS After exposure to osmium tetroxide, both healthy and diseased teeth showed a similar pattern, consisting of an unstained zone, delimited by dark stained regions, within which weakly stained areas were observed (Figs. 1 and 2). The coronal border of this zone on the healthy teeth was delimited by the gingival margin plaque (Fig. 1) and on the CAPT by the apical plaque border (Fig. 2). Root-wards, too, the zone was continuous around the entire tooth surface and ended short of the coronal remnants of the periodontal ligament fibers (Figs. 1 and 2).
SEM of Healthy Teeth At the SEM level, 4 separate regions were observed on the healthy teeth. The most coronal consisted of the gingival margin border of supragingival plaque, apical to which was an apparently plaque-free area, and then the epithelium, which corresponded to the weakly staining zone described above. The most apical comprised the coronal border of the residual periodontal ligament fibers (Fig. 3). The supragingival plaque border was usually relatively bulky, terminating where the so-called "plaque-free" zone began (Fig. 3). At the apical border cocci, rods, and filamentous bacteria predominated, but spirochetes were also observed (Fig. 4). In advance of this border, the so-called
*Prima, Byfleet, UK. 'Leica, Cambridge, UK.
"Hitachi Scientific Instruments, Berkshire, UK.
891
=
has been
stained with osmium tetroxide. A
weakly stained zone is evident below the apical plaque border (arrows), (apb apical plaque border). =
"plaque-free" zone was observed (Fig. 4). By comparison, this zone was relatively free of microorganisms, although isolated or small groups of bacteria were invariably present, chains of cocci or short rods being the predominant morphotypes (Fig. 5). Apically the area was delimited by the coronal border of the junctional epithelium, demarcating the bottom of the healthy sulcus before extraction (Fig. 3). The epithelium formed a flat sheet-like layer and corresponded to the lightly stained zone in Figure 1. It extended from the apical limit of the so-called "plaque-free" zone and formed a continuous layer extending towards the coronal border of the residual periodontal ligament fibers (Fig. 3). Isolated microorganisms were observed either close to or in contact with the most coronal epithelial cells (Fig. 6). The distance from the apical plaque border to the first attachment epithelial cells ranged form 70 to 900 µ , and from the apical plaque border to the residual ligament fibers from 1.3 to 4 mm, and the former was invariably narrower than the epithelial layer. SEM of Chronic Adult Periodontitis-Affected Teeth The diseased teeth showed a tissue zonation very similar to that of the healthy specimens, although on the root surface. The coronal margin of the epithelial layer replicated the
892
"PLAQUE-FREE ZONE"
Figure 3. SEM. Healthy extracted tooth. Note the supragingival plaque (sp) which terminates at the edge of the so-called "plaque-free" zone (arrows). Note the junctional epithelium (e) delimiting the healthy sulcus. (L residual periodontal ligament; ca contact area; pfz "plaquefree" zone; original magnification x 16; bar 1 mm). =
=
=
=
apical plaque border on all specimens examined, delimiting a middle zone (Fig. 7). The subgingival apical plaque border was similar to that described in health, tapering, or terminating in a bulky edge. The plaque border was discrete, and the bulk of the plaque did not extend into the middle zone (Fig. 7). At this border, unlike the situation on the healthy specimens, spirochetes were the most commonly observed morphotype (Fig. 8). Although at low magnification the middle zone appeared to be plaque free (Fig. 7), at higher magnifications microorganisms, though sparse, were found to be randomly dispersed throughout the area. Short rods, filaments, and spirochetes were evident, sometimes forming scattered small aggregates (Fig. 9). Compared with healthy teeth, the distance on CAPT between the subgingival apical plaque border and the epithelium was often reduced, even though this region with few organisms was a consistent feature of all specimens examined (Fig. 10). The distances from the subgingival apical plaque border to the most coronal attachment epithelial cells ranged from 40 to 600 µ and to the most coronal ligament contour
J Periodontol November 1992
IN HEALTH AND DISEASE
of the
fibers from 0.4 to 2.7 mm. The former was, as in health, always narrower than the epithelial layer. Organisms were always present in this zone, regardless of its width. A dental cuticle was discernible over the exposed cementum surface in the so-called "plaque-free" zone, as a
Figure 4. SEM. Healthy specimen showing the apical border of gingival margin plaque. Cocci (c), rods (r), filaments (f), and spirochetes (s) can be observed, (pfz plaque-free zone; original magnification x 7,000; bar 1 µ/ ). =
=
relatively smooth organic layer (Figs. 10 and 11). In some areas, the exposed cementum surface at this middle zone appeared smooth (Fig. 10), but most commonly it had uneven undulations with varying size and shape (Fig. 11). Apically, the cementum was covered by a layer of epithelium attached to its surface (Figs. 7, 10 and 12), and isolated epithelial cells were not observed within the so-called "plaque-free" zone or over the apical plaque border (Figs. 8 and 10). However, on occasions, isolated microorganisms were
noted close to
or
in contact with the most coronal
aspect of the epithelium (Fig. 12). DISCUSSION The study of the surface features of extracted teeth in relation to the pathology of chronic inflammatory periodontal disease has been widely accepted since Waerhaug14 demonstrated that, when a periodontitis-affected tooth is extracted, epithelial cells and ligament fibers remain on the tooth surface, giving information about the pathological process at the moment of the extraction. In this regard, SEM has been shown to be a useful tool as it facilitates the study of plaque, periodontal tissues, and the tooth surface itself, providing high resolution topographical images with good depth of field.15 The present study has verified the existence, on periodontitis-affected teeth, of a zone with relatively fewer or-
Volume 63 Number 11
FRIEDMAN, BARBER, MORDAN,
Figure 5. SEM. Area within the so-called "plaque-free" zone of a healthy tooth showing groups of microorganisms, (c cocci; r rods; f filaments; original magnification x 12,000; bar 1 µ/ ). =
=
=
NEWMAN
893
microprocesses (blebs) on the surface (arrow heads), (pfz 2 µ ). x 7,000; bar "plaque-free" zone; original magnification
celh show
=
=
=
=
junctional or attachment epithelium is attached, not apposed, to the tooth surface. In fact, the so-called "plaquefree" zone or zone of plaque inhibition described by Brady3 was observed in this study on all specimens of both groups as a discrete region between the most apical border of the plaque and the most coronal layer of junctional or attachment epithelial cells that remained on the tooth surface after as
ganisms than the established plaque between the most apical plaque border and the most coronal residual junctional epithelial cells, analogous to the middle, cuticular zone on children's healthy teeth.2,16 It is possible that similar zonation occurs in health in adults, although obvious ethical reasons preclude verification. Similar problems hinder investigation of healthy adult teeth where the attachment has shifted onto cementum. Therefore, this study used as controls only premolars extracted for orthodontic reasons. On periodontitis-affected extracted teeth, Waerhaug14,1719 observed that, regardless of disease level, there was a clear gap between the apical border plaque and the most coronal residual ligament fibers. The same area was analyzed by Saglie et al.8 who used the term plaque-free zone and described epithelial remnants within the zone. Newman4 also observed a middle non-staining zone at the same location and further stated it persisted on the root surface of CAPT throughout the disease process. Waerhaug18,19 suggested that this zone had been covered in life by what he called "the epithelial cuff." A recent SEM study also postulated that, in vivo, the plaque-free zone on periodontitis-affected teeth is apparently covered by a layer of cells, although they described this as junctional epithelium.13 However a more correct description would be sulcular,
extraction. It seems reasonable, therefore, to suggest that the middle zone is delimited apically by epithelium. As a corollary, the zone does not extend from the plaque to the
periodontal ligament remnants as previously described,7"9 comprises a more limited region, narrower than the epithelial layer. Saglie et al.8 asserted that the width of the zone decreased as pocket depth and loss of attachment increased. However, another study4 concluded that the zone width was indepenbut
dent of its distance from the root apex. From the present study, plaque and epithelium would appear to be generally only a small distance apart in both healthy and diseased groups. Brady3 described the average distance from plaque to epithelium as 80 µ on healthy specimens and 140 to 440 µ on periodontitis-affected teeth. The wider range presented in our study is because we considered both narrowest and widest regions. Nevertheless, there is no doubt that the distance from the plaque to the junctional epithelial cells is very narrow when compared to the distance from
894
J Periodontol November 1992
"PLAQUE-FREE ZONE" IN HEALTH AND DISEASE
Figure 7. SEM. Periodontitis-affected tooth showing the "plaque-free" (pfz) delimited coronally by the apical plaque border (arrow) and apically by the functional epithelium (e) (apb apical plaque border; L residual periodontal ligament; original magnification 25; bar 1 mm).
Figure 9. SEM. Area within the so-called "plaque-free" zone of a periodontitis-affected tooth showing a small aggregate of microorganisms (r rods;f filaments; s spirochetes; c cocci; original magnification
Figure 8. SEM. Apical plaque border spirochetes (arrow heads) (pfz magnification x 9,000; bar 1 µ ).
the plaque to the ligament fiber remnants. The larger zone widths reported in other studies7 9 were measured on extracted teeth using enlarged photographs and stereomicroscopy. At low magnification, it is difficult to determine the presence of the epithelial cells,20 and this is probably why the term plaque-free zone has persisted in relation to the whole relatively non-staining zone. It may be noted that on the present study bacteria were present in the zone regardless of its width. Regarding the bacterial morphotypes observed, the predominance of cocci and short rods at the healthy apical plaque border and of spirochetes at the diseased one, has also been previously observed.2-21 The so-called "plaquefree" zone on CAPT was not free from bacteria, again as noted previously4*22 when scattered microorganisms or small aggregates were observed extending beyond the border plaque, the most apically located being cocci and rods, with occasional filaments and spirochetes. This study did not thoroughly analyze the dental cuticle, although this structure has been previously observed by transmission electron microscopy3 and SEM20*23 in this middle zone and beneath the epithelial cells and is thought to derive from adsorption of components of the gingival exúdate to the root surface.5*6 The smooth appearance of the cementum surface in some areas of the CAPT teeth may be attributed
zone
=
=
=
many
=
in =
a
diseased
specimen showing zone; original
"plaque-free"
=
x
=
12,000;
bar
=
=
1
=
µ ).
Volume 63
Number 11
FRIEDMAN, BARBER, MORDAN,
NEWMAN
895
11. SEM showing the organic layer of dental cuticle covering the undulated cementum surface at the "plaque-free" zone (original magnification 1,000; bar 0.1 µ ).
Figure
=
Figure 10. SEM of a diseased tooth. Note the smooth cementum covered by an organic layer, the dental cuticle, in this area of the so-called "plaquefree" zone (arrow) (e epithelium;pfz "plaque-free"zone; original magnification 900; bar 5 µ ). =
=
=
to this organic covering.3,20,23,24 In other areas the cuticle covered the rounded elevations of the cementum obscuring the surface detail. These appear to correspond to where Sharpey fiber bundles were inserted previous to the apical migration of the junctional epithelium.15,25 Although occasional leukocytes were observed, the absence of a polymorphonuclear neutrophil (PMN) layer as such at the so-called "plaque-free" zone, and the presence of only isolated microorganisms close to the junctional epithelium on the cementum surface described here, may be explained by true absence of adhering bacteria or lack of firm attachment and removal during extraction or the washing procedure.11,22 However, microorganisms can be extremely close to epithelium in this region, as may be deduced from block sections where there is clear evidence of lack of disturbance,26,27 although in such instances there is usually artificial interference with the host response, for example, depletion of PMN. Therefore, on these extracted
Figure 12. SEM. Periodontitis-affected tooth showing the "plaque-free" (pfz) delimited apically by junctional epithelium (e). Few microorganisms are observed both on the cementum surface (black arrow) and on the epithelial surface (white arrow heads) (original magnification zone
3,000; bar
=
0.5 µ ).
human teeth,
epithelium
microorganisms in middle zone and close to likely to reflect the in vivo situation. How-
are
ever, abundant bacteria and PMN cannot be considered
as
major structural elements of this middle zone. In summary, this study has confirmed that the tissue zonation observed on the enamel surfaces of healthy extracted
teeth is maintained
on
the root surfaces of chronic adult
896
"PLAQUE-FREE ZONE"
J Periodontol November 1992
IN HEALTH AND DISEASE
periodontitis-affected teeth. The middle zone was observed on all specimens of both groups as an area covered by a cuticular layer and with few bacteria between the apical border of the supra- or subgingival plaque respectively and the most coronal level of the epithelial cells. The zone between the apical plaque border and the periodontal ligament fibers should not continue to be described as plaquefree as bacteria were always present in this region. The findings further suggest that a region of lining epithelium just coronal to the bottom of the periodontal pocket, analogous to the sulcular epithelium on healthy teeth, is firmly apposed but not organically attached to the root before extraction, and prevents the bulk of the plaque from reaching the junctional epithelium. It is concluded that a tissue zonation resembling that in health on enamel, though of different origin, comprising a discrete plaque border, a middle zone covered by dental cuticle, where sparse organisms are observed, and apically delimited by epithelium, is evident on the root surfaces of chronic adult periodontitis-affected teeth throughout the disease process. It is postulated that this tissue complex has a protective role during the entire course of chronic inflammatory periodontal disease.
8.
9. Powell
REFERENCES 1. Bass CC. A demonstrable line on extracted teeth indicating the location of the outer border of the epithelial attachment. / Dent Res 1946; 25:40L415. 2. Newman HN. Zone demarcation of organic films present on human enamel surfaces in vivo. Brit Dent J 1973; 134:273-278. 3. Brady JM. A plaque-free zone on human teeth—scanning and transmission electron microscopy. J Periodontol 1973; 44:416-428. 4. Newman HN. The apical border of plaque in chronic inflammatory periodontal disease. Brit Dent J 1976; 141:105-113. 5. Eide , Lie , Selvig KA. Surface coatings on dental cementum incident to periodontal disease. I. A scanning electron microscope study. / Clin Periodontol 1983; 10:157-171. 6. Eide , Lie , Selvig KA. Surface coatings on dental cementum incident to periodontal disease. II. A scanning electron microscopic confirmation of a mineralized cuticle. / Clin Periodontol 1984; 11:565-575. 7. Hoffman ID, Gold W. Distances between plaque and remnants of attached periodontal tissues on extracted teeth. J Periodontol 1971; 42:29-30.
B, Garnick JJ. The
measurements of
of extracted teeth to evaluate clinical disease. / Periodontol 1978; 49:621-
use
periodontal
624. 10.
11.
12.
13.
14.
15.
16.
17.
18.
Acknowledgments
We thank Professor M. Harris and his colleagues of the Department of Maxillo-Facial Surgery and Oral Medicine for their help in obtaining the extracted teeth. We are indebted to the Electron Microscopy Unit of the British Museum of Natural History for the use of their S-800 Hitachi field emission scanning electron microscope. We acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnològico (CNPq)-Brazil for the support of M.T. Friedman.
Saglie R, Johansen JR, Tollefsen T. Plaque-free zones on human teeth in Periodontitis. J Clin Periodontol 1975; 2:190-197.
19.
20.
21.
22.
23. 24.
Vrahopoulos TP, Barber P, Liakoni H, Newman HN. Ultrastructure of the periodontal lesion in a case of Papillon-Lefèvre syndrome (PLS). JClin Periodontol 1988; 15:17-26. Douglass KD, Cobb CM, Berkstein S, Killoy WJ. Microscopic characterization of root surface-associated microbial plaque in localized juvenile Periodontitis. / Periodontol 1990; 60:475-484. Garnick JJ, Dent J. A scanning electron micrographical study of root surfaces and subgingival bacteria after hand and ultrasonic instrumentation. J Periodontol 1989; 60:441-147. Cobb CM, Killoy WJ. Microbial colonization in human periodontal disease: An illustrated tutorial on selected ultrastructural and ecologie considerations. Scanning Microsc 1990; 4:675-691. Waerhaug J. A method for evaluation of periodontal problems on extracted teeth. J Clin Periodontol 1975; 2:160-168. Carrassi A, Abati S, Santarelli G. The role of scanning electron microscopy in periodontal research. Scanning Microsc 1988; 2:11231138. Newman HN. Ultrastructure of the apical border of dental plaque. In: The Borderland Between Caries and Periodontal Disease. Lehner T, ed. London: Academic Press; 1977: 79-103. Waerhaug J. The gingival pocket. Anatomy, pathology, deepening and elimination. Odontol Tds 1952; 60(suppl. 1):117-134. Waerhaug J. Subgingival plaque and loss of attachment in periodontosis as evaluated on extracted teeth. / Periodontol 1977; 48:125130. Waerhaug J. Healing of the dento-epithelial junction following subgingival plaque control. II. As observed on extracted teeth. J Periodontol 1978; 49:119-134. Garnick JJ, Ringle RD. The dento-gingival junction as seen with light microscopy and scanning electron microscopy. Scanning Microsc 1988; 2:1113-1122. Newman HN. The approximal apical border of plaque on children's teeth 1. Morphology, structure and cell content. J Periodontol 1979; 50:561-567. Saglie RA. A scanning electron microscopy study of the relationship between the most apically located subgingival plaque and the epithelial attachment. J Periodontol 1977; 48:105-115. Jones SJ. The tooth surface in periodontal disease. Dent Practit 1972; 22:462-473. Schroeder HE, Listgarten MA. Fine Structure of Developing Epithelial Attachment of Human Teeth; 2nd ed. Basel: S. Karger; 1977:91-
96. 25. Jones SJ. The root surface: An illustrated review of some scanning electron microscope studies. Scanning Microsc 1987; 1:2003-2018. 26. Schroeder HE, Attström R. Pocket formation: An hypothesis. In: The Borderland Between Caries and Periodontal Disease II. Lehner T, Cimasoni C, eds. London: Academic Press; 1980: 99-123. 27. Theilade J, Attström R. Distribution and ultrastructure of subgingival plaque in beagle dogs with gingival inflammation. J Periodont Res 1985; 20:131-145.
Send reprint requests to: Dr. M.T. Friedman, Institute of Dental Surgery, Eastman's Dental Hospital, 256 Gray's Inn Road, London WC1X 8LD UK. Accepted for publication May 8, 1992.
The "Plaque-Free Zone" in Health and Disease: A Scanning Electron
Microscope Study Marlize T. Friedman, * Pauline M. Barber, * Nicola J. Mordan, * and Hubert N.
Newman*f
A "plaque-free" zone has been described on the enamel surface of healthy extracted teeth. This study examined this zone on chronic adult periodontitis-affected teeth (CAPT). Ten healthy controls and 16 CAPT were collected immediately after extraction, fixed, dehydrated, critical point dried, coated, and viewed by scanning electron microscopy (SEM). The "plaque-free" zone was observed in both groups as an area with few bacteria between the apical plaque border and the coronal limit of an epithelial layer on the root surface, extending to the residual periodontal ligament. On the healthy specimens, the apical plaque border consisted mainly of cocci and short rods, while on the CAPT specimens spirochetes predominated. Isolated or small groups of microorganisms were always present in the "plaque-free" zone and at its apical limit, close to or in contact with junctional epithelial cells. This zone is therefore not completely free of plaque, as suggested. It was concluded that a tissue complex, analogous to that in health on enamel, persists on the root surfaces of CAPT throughout the disease process. It comprises a discrete plaque border, a dental cuticle with sparse organisms, and an epithelium analogous to junctional epithelium. Its main function would appear to be to prevent bulk access of plaque to the surrounding tissues, including direct contact of bacteria with underlying ligament. / Periodontology 1992; 63:890-896.
Key Words: Dental plaque/epidemiology; dental plaque/microbiology; periodontitis/mi-
crobiology.
A non-staining zone is a common finding on the smooth surfaces of both healthy and diseased, stained, extracted teeth. It was first described by Bass.1 On the enamel of healthy teeth, this zone is delimited coronally by the gingival margin plaque and apically by the junctional epithelium. It corresponds, corono-apically, to the healthy crevice before extraction, and microorganisms are sparse in this
region.2 In
1973, Brady3 used scanning and transmission electron
microscopy to examine this zone on both healthy and periodontitis-affected teeth. He, too, described it as an area virtually free from microorganisms, between the apical plaque border and the attachment epithelium, and called it the "plaque-free zone" (PFZ). Although there is agreement regarding the principal structural components of this zone on enamel, there is little knowledge concerning its features on diseased teeth. While it is generally accepted that on the latter the coronal limit is the established subgingival plaque 'Electron
Microscopy Unit,
Institute of Dental
London, London UK.
Department of Periodontology.
Surgery, University
of
border4 and that the underlying cementum is covered by a dental cuticle,5'6 most views differ from Brady's description of its apical limit. On periodontitis-affected teeth, the PFZ apical limit has been described as the coronal border of the residual periodontal ligament fibers.712 Recently, however, another study13 has also suggested that, in vivo, the zone is apically limited by epithelial cells. Although this zone is now always referred to as the plaquefree zone and this term is currently accepted, its apical limit on the root surface of diseased teeth is not yet clear and the designation of plaque-free requires investigation. The aims of the present study, therefore, were to identify the components of this specific zone of the root surface of chronic adult periodontitis-affected teeth (CAPT). MATERIALS AND METHODS The material for this study comprised 10 healthy and 16 chronic adult periodontitis-affected human teeth (CAPT), collected immediately after extraction from patients referred to the Department of Maxillo-Facial Surgery and Oral Medicine. Consent was obtained from all patients, or their guardians in the case of children, concerning the study of
Volume 63 Number 11
their extracted teeth. Healthy teeth, extracted for orthodontic reasons, were obtained from patients aged 12 to 14 years, and the diseased teeth from patients aged 35 to 65 years. The inclusion criteria for CAPT were: no history of periodontal treatment for at least 1 year prior to extraction, bleeding on gentle probing, pocket depth >4 mm in relation to at least one of the approximal surfaces, and radiographie evidence of bone loss. Clinical examination was always performed by the same operator (MTF). Pocket depth was measured to the nearest millimeter with a Borodontic probe* and a Williams tip (diameter 0.5 mm), calibrated at 0.25N. All teeth were extracted without approximal instrumentation and immediately rinsed in 0.1M sodium cacodylate buffer solution (pH 7.4) to remove blood and loose debris. They were then fixed in 3% glutaraldehyde in 0.1M sodium cacodylate buffer, previously cooled at 4°C, for 3 hours. After primary fixation, teeth were post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 hours at 4°C. They were dehydrated in a graded aqueous acetone series, critical-point dried, sputter-coated with gold-palladium and viewed in a scanning electron microscope*5 or a field emission scanning electron microscope.11 The distances from the apical plaque border to the most coronal attachment epithelial cells and from the plaque border to the residual periodontal ligament fibers were measured on scanning electron micrographs.
FRIEDMAN, BARBER, MORDAN, NEWMAN
1. (Left) A healthy extracted tooth that has been stained with osmium tetroxide. Note the weakly stained band within the unstained zone
Figure
(arrows), (gm gingival margin plaque; ca contact area). Figure 2. (Right) A periodontally-affected extracted tooth that =
RESULTS After exposure to osmium tetroxide, both healthy and diseased teeth showed a similar pattern, consisting of an unstained zone, delimited by dark stained regions, within which weakly stained areas were observed (Figs. 1 and 2). The coronal border of this zone on the healthy teeth was delimited by the gingival margin plaque (Fig. 1) and on the CAPT by the apical plaque border (Fig. 2). Root-wards, too, the zone was continuous around the entire tooth surface and ended short of the coronal remnants of the periodontal ligament fibers (Figs. 1 and 2).
SEM of Healthy Teeth At the SEM level, 4 separate regions were observed on the healthy teeth. The most coronal consisted of the gingival margin border of supragingival plaque, apical to which was an apparently plaque-free area, and then the epithelium, which corresponded to the weakly staining zone described above. The most apical comprised the coronal border of the residual periodontal ligament fibers (Fig. 3). The supragingival plaque border was usually relatively bulky, terminating where the so-called "plaque-free" zone began (Fig. 3). At the apical border cocci, rods, and filamentous bacteria predominated, but spirochetes were also observed (Fig. 4). In advance of this border, the so-called
*Prima, Byfleet, UK. 'Leica, Cambridge, UK.
"Hitachi Scientific Instruments, Berkshire, UK.
891
=
has been
stained with osmium tetroxide. A
weakly stained zone is evident below the apical plaque border (arrows), (apb apical plaque border). =
"plaque-free" zone was observed (Fig. 4). By comparison, this zone was relatively free of microorganisms, although isolated or small groups of bacteria were invariably present, chains of cocci or short rods being the predominant morphotypes (Fig. 5). Apically the area was delimited by the coronal border of the junctional epithelium, demarcating the bottom of the healthy sulcus before extraction (Fig. 3). The epithelium formed a flat sheet-like layer and corresponded to the lightly stained zone in Figure 1. It extended from the apical limit of the so-called "plaque-free" zone and formed a continuous layer extending towards the coronal border of the residual periodontal ligament fibers (Fig. 3). Isolated microorganisms were observed either close to or in contact with the most coronal epithelial cells (Fig. 6). The distance from the apical plaque border to the first attachment epithelial cells ranged form 70 to 900 µ , and from the apical plaque border to the residual ligament fibers from 1.3 to 4 mm, and the former was invariably narrower than the epithelial layer. SEM of Chronic Adult Periodontitis-Affected Teeth The diseased teeth showed a tissue zonation very similar to that of the healthy specimens, although on the root surface. The coronal margin of the epithelial layer replicated the
892
"PLAQUE-FREE ZONE"
Figure 3. SEM. Healthy extracted tooth. Note the supragingival plaque (sp) which terminates at the edge of the so-called "plaque-free" zone (arrows). Note the junctional epithelium (e) delimiting the healthy sulcus. (L residual periodontal ligament; ca contact area; pfz "plaquefree" zone; original magnification x 16; bar 1 mm). =
=
=
=
apical plaque border on all specimens examined, delimiting a middle zone (Fig. 7). The subgingival apical plaque border was similar to that described in health, tapering, or terminating in a bulky edge. The plaque border was discrete, and the bulk of the plaque did not extend into the middle zone (Fig. 7). At this border, unlike the situation on the healthy specimens, spirochetes were the most commonly observed morphotype (Fig. 8). Although at low magnification the middle zone appeared to be plaque free (Fig. 7), at higher magnifications microorganisms, though sparse, were found to be randomly dispersed throughout the area. Short rods, filaments, and spirochetes were evident, sometimes forming scattered small aggregates (Fig. 9). Compared with healthy teeth, the distance on CAPT between the subgingival apical plaque border and the epithelium was often reduced, even though this region with few organisms was a consistent feature of all specimens examined (Fig. 10). The distances from the subgingival apical plaque border to the most coronal attachment epithelial cells ranged from 40 to 600 µ and to the most coronal ligament contour
J Periodontol November 1992
IN HEALTH AND DISEASE
of the
fibers from 0.4 to 2.7 mm. The former was, as in health, always narrower than the epithelial layer. Organisms were always present in this zone, regardless of its width. A dental cuticle was discernible over the exposed cementum surface in the so-called "plaque-free" zone, as a
Figure 4. SEM. Healthy specimen showing the apical border of gingival margin plaque. Cocci (c), rods (r), filaments (f), and spirochetes (s) can be observed, (pfz plaque-free zone; original magnification x 7,000; bar 1 µ/ ). =
=
relatively smooth organic layer (Figs. 10 and 11). In some areas, the exposed cementum surface at this middle zone appeared smooth (Fig. 10), but most commonly it had uneven undulations with varying size and shape (Fig. 11). Apically, the cementum was covered by a layer of epithelium attached to its surface (Figs. 7, 10 and 12), and isolated epithelial cells were not observed within the so-called "plaque-free" zone or over the apical plaque border (Figs. 8 and 10). However, on occasions, isolated microorganisms were
noted close to
or
in contact with the most coronal
aspect of the epithelium (Fig. 12). DISCUSSION The study of the surface features of extracted teeth in relation to the pathology of chronic inflammatory periodontal disease has been widely accepted since Waerhaug14 demonstrated that, when a periodontitis-affected tooth is extracted, epithelial cells and ligament fibers remain on the tooth surface, giving information about the pathological process at the moment of the extraction. In this regard, SEM has been shown to be a useful tool as it facilitates the study of plaque, periodontal tissues, and the tooth surface itself, providing high resolution topographical images with good depth of field.15 The present study has verified the existence, on periodontitis-affected teeth, of a zone with relatively fewer or-
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FRIEDMAN, BARBER, MORDAN,
Figure 5. SEM. Area within the so-called "plaque-free" zone of a healthy tooth showing groups of microorganisms, (c cocci; r rods; f filaments; original magnification x 12,000; bar 1 µ/ ). =
=
=
NEWMAN
893
microprocesses (blebs) on the surface (arrow heads), (pfz 2 µ ). x 7,000; bar "plaque-free" zone; original magnification
celh show
=
=
=
=
junctional or attachment epithelium is attached, not apposed, to the tooth surface. In fact, the so-called "plaquefree" zone or zone of plaque inhibition described by Brady3 was observed in this study on all specimens of both groups as a discrete region between the most apical border of the plaque and the most coronal layer of junctional or attachment epithelial cells that remained on the tooth surface after as
ganisms than the established plaque between the most apical plaque border and the most coronal residual junctional epithelial cells, analogous to the middle, cuticular zone on children's healthy teeth.2,16 It is possible that similar zonation occurs in health in adults, although obvious ethical reasons preclude verification. Similar problems hinder investigation of healthy adult teeth where the attachment has shifted onto cementum. Therefore, this study used as controls only premolars extracted for orthodontic reasons. On periodontitis-affected extracted teeth, Waerhaug14,1719 observed that, regardless of disease level, there was a clear gap between the apical border plaque and the most coronal residual ligament fibers. The same area was analyzed by Saglie et al.8 who used the term plaque-free zone and described epithelial remnants within the zone. Newman4 also observed a middle non-staining zone at the same location and further stated it persisted on the root surface of CAPT throughout the disease process. Waerhaug18,19 suggested that this zone had been covered in life by what he called "the epithelial cuff." A recent SEM study also postulated that, in vivo, the plaque-free zone on periodontitis-affected teeth is apparently covered by a layer of cells, although they described this as junctional epithelium.13 However a more correct description would be sulcular,
extraction. It seems reasonable, therefore, to suggest that the middle zone is delimited apically by epithelium. As a corollary, the zone does not extend from the plaque to the
periodontal ligament remnants as previously described,7"9 comprises a more limited region, narrower than the epithelial layer. Saglie et al.8 asserted that the width of the zone decreased as pocket depth and loss of attachment increased. However, another study4 concluded that the zone width was indepenbut
dent of its distance from the root apex. From the present study, plaque and epithelium would appear to be generally only a small distance apart in both healthy and diseased groups. Brady3 described the average distance from plaque to epithelium as 80 µ on healthy specimens and 140 to 440 µ on periodontitis-affected teeth. The wider range presented in our study is because we considered both narrowest and widest regions. Nevertheless, there is no doubt that the distance from the plaque to the junctional epithelial cells is very narrow when compared to the distance from
894
J Periodontol November 1992
"PLAQUE-FREE ZONE" IN HEALTH AND DISEASE
Figure 7. SEM. Periodontitis-affected tooth showing the "plaque-free" (pfz) delimited coronally by the apical plaque border (arrow) and apically by the functional epithelium (e) (apb apical plaque border; L residual periodontal ligament; original magnification 25; bar 1 mm).
Figure 9. SEM. Area within the so-called "plaque-free" zone of a periodontitis-affected tooth showing a small aggregate of microorganisms (r rods;f filaments; s spirochetes; c cocci; original magnification
Figure 8. SEM. Apical plaque border spirochetes (arrow heads) (pfz magnification x 9,000; bar 1 µ ).
the plaque to the ligament fiber remnants. The larger zone widths reported in other studies7 9 were measured on extracted teeth using enlarged photographs and stereomicroscopy. At low magnification, it is difficult to determine the presence of the epithelial cells,20 and this is probably why the term plaque-free zone has persisted in relation to the whole relatively non-staining zone. It may be noted that on the present study bacteria were present in the zone regardless of its width. Regarding the bacterial morphotypes observed, the predominance of cocci and short rods at the healthy apical plaque border and of spirochetes at the diseased one, has also been previously observed.2-21 The so-called "plaquefree" zone on CAPT was not free from bacteria, again as noted previously4*22 when scattered microorganisms or small aggregates were observed extending beyond the border plaque, the most apically located being cocci and rods, with occasional filaments and spirochetes. This study did not thoroughly analyze the dental cuticle, although this structure has been previously observed by transmission electron microscopy3 and SEM20*23 in this middle zone and beneath the epithelial cells and is thought to derive from adsorption of components of the gingival exúdate to the root surface.5*6 The smooth appearance of the cementum surface in some areas of the CAPT teeth may be attributed
zone
=
=
=
many
=
in =
a
diseased
specimen showing zone; original
"plaque-free"
=
x
=
12,000;
bar
=
=
1
=
µ ).
Volume 63
Number 11
FRIEDMAN, BARBER, MORDAN,
NEWMAN
895
11. SEM showing the organic layer of dental cuticle covering the undulated cementum surface at the "plaque-free" zone (original magnification 1,000; bar 0.1 µ ).
Figure
=
Figure 10. SEM of a diseased tooth. Note the smooth cementum covered by an organic layer, the dental cuticle, in this area of the so-called "plaquefree" zone (arrow) (e epithelium;pfz "plaque-free"zone; original magnification 900; bar 5 µ ). =
=
=
to this organic covering.3,20,23,24 In other areas the cuticle covered the rounded elevations of the cementum obscuring the surface detail. These appear to correspond to where Sharpey fiber bundles were inserted previous to the apical migration of the junctional epithelium.15,25 Although occasional leukocytes were observed, the absence of a polymorphonuclear neutrophil (PMN) layer as such at the so-called "plaque-free" zone, and the presence of only isolated microorganisms close to the junctional epithelium on the cementum surface described here, may be explained by true absence of adhering bacteria or lack of firm attachment and removal during extraction or the washing procedure.11,22 However, microorganisms can be extremely close to epithelium in this region, as may be deduced from block sections where there is clear evidence of lack of disturbance,26,27 although in such instances there is usually artificial interference with the host response, for example, depletion of PMN. Therefore, on these extracted
Figure 12. SEM. Periodontitis-affected tooth showing the "plaque-free" (pfz) delimited apically by junctional epithelium (e). Few microorganisms are observed both on the cementum surface (black arrow) and on the epithelial surface (white arrow heads) (original magnification zone
3,000; bar
=
0.5 µ ).
human teeth,
epithelium
microorganisms in middle zone and close to likely to reflect the in vivo situation. How-
are
ever, abundant bacteria and PMN cannot be considered
as
major structural elements of this middle zone. In summary, this study has confirmed that the tissue zonation observed on the enamel surfaces of healthy extracted
teeth is maintained
on
the root surfaces of chronic adult
896
"PLAQUE-FREE ZONE"
J Periodontol November 1992
IN HEALTH AND DISEASE
periodontitis-affected teeth. The middle zone was observed on all specimens of both groups as an area covered by a cuticular layer and with few bacteria between the apical border of the supra- or subgingival plaque respectively and the most coronal level of the epithelial cells. The zone between the apical plaque border and the periodontal ligament fibers should not continue to be described as plaquefree as bacteria were always present in this region. The findings further suggest that a region of lining epithelium just coronal to the bottom of the periodontal pocket, analogous to the sulcular epithelium on healthy teeth, is firmly apposed but not organically attached to the root before extraction, and prevents the bulk of the plaque from reaching the junctional epithelium. It is concluded that a tissue zonation resembling that in health on enamel, though of different origin, comprising a discrete plaque border, a middle zone covered by dental cuticle, where sparse organisms are observed, and apically delimited by epithelium, is evident on the root surfaces of chronic adult periodontitis-affected teeth throughout the disease process. It is postulated that this tissue complex has a protective role during the entire course of chronic inflammatory periodontal disease.
8.
9. Powell
REFERENCES 1. Bass CC. A demonstrable line on extracted teeth indicating the location of the outer border of the epithelial attachment. / Dent Res 1946; 25:40L415. 2. Newman HN. Zone demarcation of organic films present on human enamel surfaces in vivo. Brit Dent J 1973; 134:273-278. 3. Brady JM. A plaque-free zone on human teeth—scanning and transmission electron microscopy. J Periodontol 1973; 44:416-428. 4. Newman HN. The apical border of plaque in chronic inflammatory periodontal disease. Brit Dent J 1976; 141:105-113. 5. Eide , Lie , Selvig KA. Surface coatings on dental cementum incident to periodontal disease. I. A scanning electron microscope study. / Clin Periodontol 1983; 10:157-171. 6. Eide , Lie , Selvig KA. Surface coatings on dental cementum incident to periodontal disease. II. A scanning electron microscopic confirmation of a mineralized cuticle. / Clin Periodontol 1984; 11:565-575. 7. Hoffman ID, Gold W. Distances between plaque and remnants of attached periodontal tissues on extracted teeth. J Periodontol 1971; 42:29-30.
B, Garnick JJ. The
measurements of
of extracted teeth to evaluate clinical disease. / Periodontol 1978; 49:621-
use
periodontal
624. 10.
11.
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13.
14.
15.
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17.
18.
Acknowledgments
We thank Professor M. Harris and his colleagues of the Department of Maxillo-Facial Surgery and Oral Medicine for their help in obtaining the extracted teeth. We are indebted to the Electron Microscopy Unit of the British Museum of Natural History for the use of their S-800 Hitachi field emission scanning electron microscope. We acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnològico (CNPq)-Brazil for the support of M.T. Friedman.
Saglie R, Johansen JR, Tollefsen T. Plaque-free zones on human teeth in Periodontitis. J Clin Periodontol 1975; 2:190-197.
19.
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23. 24.
Vrahopoulos TP, Barber P, Liakoni H, Newman HN. Ultrastructure of the periodontal lesion in a case of Papillon-Lefèvre syndrome (PLS). JClin Periodontol 1988; 15:17-26. Douglass KD, Cobb CM, Berkstein S, Killoy WJ. Microscopic characterization of root surface-associated microbial plaque in localized juvenile Periodontitis. / Periodontol 1990; 60:475-484. Garnick JJ, Dent J. A scanning electron micrographical study of root surfaces and subgingival bacteria after hand and ultrasonic instrumentation. J Periodontol 1989; 60:441-147. Cobb CM, Killoy WJ. Microbial colonization in human periodontal disease: An illustrated tutorial on selected ultrastructural and ecologie considerations. Scanning Microsc 1990; 4:675-691. Waerhaug J. A method for evaluation of periodontal problems on extracted teeth. J Clin Periodontol 1975; 2:160-168. Carrassi A, Abati S, Santarelli G. The role of scanning electron microscopy in periodontal research. Scanning Microsc 1988; 2:11231138. Newman HN. Ultrastructure of the apical border of dental plaque. In: The Borderland Between Caries and Periodontal Disease. Lehner T, ed. London: Academic Press; 1977: 79-103. Waerhaug J. The gingival pocket. Anatomy, pathology, deepening and elimination. Odontol Tds 1952; 60(suppl. 1):117-134. Waerhaug J. Subgingival plaque and loss of attachment in periodontosis as evaluated on extracted teeth. / Periodontol 1977; 48:125130. Waerhaug J. Healing of the dento-epithelial junction following subgingival plaque control. II. As observed on extracted teeth. J Periodontol 1978; 49:119-134. Garnick JJ, Ringle RD. The dento-gingival junction as seen with light microscopy and scanning electron microscopy. Scanning Microsc 1988; 2:1113-1122. Newman HN. The approximal apical border of plaque on children's teeth 1. Morphology, structure and cell content. J Periodontol 1979; 50:561-567. Saglie RA. A scanning electron microscopy study of the relationship between the most apically located subgingival plaque and the epithelial attachment. J Periodontol 1977; 48:105-115. Jones SJ. The tooth surface in periodontal disease. Dent Practit 1972; 22:462-473. Schroeder HE, Listgarten MA. Fine Structure of Developing Epithelial Attachment of Human Teeth; 2nd ed. Basel: S. Karger; 1977:91-
96. 25. Jones SJ. The root surface: An illustrated review of some scanning electron microscope studies. Scanning Microsc 1987; 1:2003-2018. 26. Schroeder HE, Attström R. Pocket formation: An hypothesis. In: The Borderland Between Caries and Periodontal Disease II. Lehner T, Cimasoni C, eds. London: Academic Press; 1980: 99-123. 27. Theilade J, Attström R. Distribution and ultrastructure of subgingival plaque in beagle dogs with gingival inflammation. J Periodont Res 1985; 20:131-145.
Send reprint requests to: Dr. M.T. Friedman, Institute of Dental Surgery, Eastman's Dental Hospital, 256 Gray's Inn Road, London WC1X 8LD UK. Accepted for publication May 8, 1992.
