SCANNING ELECTRON MICROSCOPY OF THE COLLAGEN FIBRE ARCHITECTURE OF THE RABBIT INCISOR PERIODONTIUM P. MRC
Dental
Unit,
SLOAN
The Dental School, Lower Maudlin Street. Bristol BSI 2LY. England
Summary-The structure of the rabbit incisor periodontium, prepared by slicing demineralised formalin-fixed jaws in various planes, was investigated. Three zones, corresponding to those recognised histologically, could be distinguished. The alveolar zone contained collagenous fibre bundles It%20 pm in diameter and large blood vessels. The collagen fibres in the middle zone were arranged in sheets which formed the structural basis of a series of radially-flattened compartments in the long axis of the tooth. Fibroblasts were often observed lying in the compartments. The collagen fibres in the cement zone were organised as bundles 3-5 pm in diameter. These appearances could be correlated with histological appearances; the arrangement of the fibres in the middle zone is in accord with current concepts of ligament remodelling.
lNTRODUCTlON
The arrangement of the collagen fibres in the periodontal ligament of continuously-erupting teeth has been the subject of controversy. On the basis of light microscope observations, Sicher (1923, 1942) described alveolar, middle and cement zones in the rat incisor and guinea-pig molar periodontal ligament, best seen in longitudinal sections. In the alveolar and cement zones, the collagen was organised as bundles which were continuous with the Sharpey fibres. The middle zone was composed of pre-collagenous fibres. Collagen fibres from the cement and alveolar zones could be traced into this zone for a short distance, forming the so-called intermediate plexus. An arrangement of the periodontal ligament into three zones was described in guinea-pig molar by Hunt (1959) and in rabbit incisor by Ness and Smale (1959). Using 0.5~pm sections, Smith and Warshawsky (1976) described an intermediate plexus in the periodontal ligament of the rat incisor. Beertsen, Everts and van den Hooff (1974) described variations in cell morphology and ultrastructure within the three zones in the rat incisor. Hindle (1967) traced fibre bundles across the periodontal ligament in the rat incisor in transverse sections but not in longitudinal sections, where the bundles were not continuous across the middle zone. Using polarizing microscopy, however, he found a reduction in the birefringence of the middle zone compared to the cement and alveolar zones in both transverse and longitudinal sections and thus favoured the concept of a middle zone composed mainly of pre-collagenous fibres. The existence of the intermediate plexus was disputed by Ciancio, Neiders and Hazen (1967) who traced fibre bundles across the periodontal ligament and considered that the appearance of an intermediate plexus was due to oblique sectioning. Sloan, Shellis and Berkovitz (1976) described effects of certain methods of specimen preparation on the appearance of rat periodontal ligament in the scanning electron microscope (SEM). Two techniques preserved the structure of the ligament; these were freeze-
fracture and slicing demineralized tissue with a razor blade, followed by critical-point drying. Because critical-point drying has the advantage that the plane of section can be controlled, it was decided to employ this method to examine the fibrous architecture of the ligament. The lower incisor of the rabbit was chosen because its size relative to the rat, for instance, permitted more accurate definition of the angulation and location of the section plane. MATERIALS
AND
METHODS
Seven young adult albino rabbits were killed by decapitation following anaesthesia with Nembutal. The mandibles were dissected out, divided in the rnidline and placed in 10 per cent neutral formal-saline. After two hours, the portions of the alveolar process containing the incisor teeth were separated from the rest of the jaw with a diamond disc and fixed for 7 days in fresh neutral formol-saline. The specimens were demineralised in 10 per cent ethylene diamine tetra-acetic acid (EDTA), pH 7.4, with continuous stirring. The solution was renewed weekly and the process continued until no mineral was detectable radiographically. After washing in 0.2 M phosphate buffer at pH 7.4 for 1 h, the incisors were serially divided into 3-mm slices with cuts transverse or oblique to the long axis of the tooth. Each slice was then divided in the median or paramedian plane (Text Fig. 1). After sectioning, all the blocks were stored in 0.2 M phosphate buffer. Some blocks which had been sliced transversely or in median planes were selected and incubated in crude bacterial r-amylase (BDH Chemicals Ltd., Poole, Dorset) at a concentration of I mg/ml in 0.2 M phosphate buffer at 37°C for 2 h to remove ground substance (Hunter and Finlay, 1973). All specimens were then washed in distilled water, dehydrated in a graded ethanol series, critical-point dried in amyl acetate from carbon dioxide, mounted on Stereoscan stubs and coated with carbon and gold/ palladium alloy. The specimens were examined in a Stereoscan 600 (Cambridge Instruments Ltd., Cam567
568
P. Sloan
Fig. 1. Section
planes
used in the SEM study.
bridge, England) at an accelerating voltage of 15 V, using a variety of tilt angles. Stereo-pair photographs of the ligament in each section plane and of the regions where two planes intersected were taken. Two pairs of demineralised lower incisors were dehydrated and embedded in paraffin wax. Serial sections were cut at 8 m in the transverse and median planes and stained with Ehrlich haematoxylineosin or Heidenhain azan trichrome. RESULTS
Histological observations Only findings which are relevant to understanding SEM appearances will be described here. In both the transverse and median planes, three zones within the periodontal ligament could be distinguished (Plate Figs. 2 and 3). The alveolar zone occupied approximately 40 per cent of the ligament width and contained the main periodontal blood vessels, which were larger and more numerous at the basal end of the tooth. The collagen fibres in the alveolar zone were organised as large bundles which coursed between the blood vessels and were inserted into the bone at approximately right angles to the mineralized surface. The fibroblasts in this region were spindle-shaped in both transverse and median sections. The middle zone occupied about 50 per cent of the width of the ligament. In transverse section, it appeared to consist of radially-orientated bundles which arose by subdivision of the larger bundles in the alveolar zone. The bundles in the middle zone branched and anastomosed with each other (Plate Fig. 2). In median section, however, the fibres of the middle zone did not appear to be organised as large bundles, but appeared as a meshwork of individual fine fibres (Plate Fig. 3). The fibroblasts in the middle zone were spindle-shaped in transverse section, but in median section had approximately circular outlines.
Median
refers to the midline
of the tooth
The cement zone occupied up to 10 per cent of the ligament width and was similar in transverse and median sections. The collagen fibres were organised as circular bundles somewhat smaller than those in the alveolar zone, which were separated by cuboidal cells. SEA4 observations In transverse sections (Plate Fig. 4) alveolar, middle and cement zones could be distinguished. The alveolar zone occupied about 40 per cent of the width of the ligament and contained bundles lC20pm in diameter, composed of collagen fibres. These were continuous with the Sharpey fibres. The middle zone appeared to consist of fibre bundles l-3 pm in diameter which were separated by a distance of 0.5-I pm and arose by branching of the larger bundles in the alveolar zone. They formed a continuous network by branching and anastomosing with each other. Branching occurred at 3%10pm intervals. In places, the bundles were wavy in the transverse plane and were always radially orientated on the medial aspect of the tooth. The orientation of the fibres in the lateral parts of the ligament changed along the length of the tooth; in the alveolar crest region, they passed straight from cement to bone but followed a progressively more oblique course towards the growing end. The bundles in the cement zone were 3-5 pm in diameter and were orientated at approximately right angles to the mineralized surface. In the median plane, the collagen in the alveolar and cement zones was also organised as bundles (Plate Fig. 5) which fanned out into the middle zone (Plate Figs. 6 and 7). In this plane, the middle zone appeared as an undulating, sheet-like continuum of fibres which formed a series of compartments. In oblique sections (Plate Fig. 8), the compartments formed by the sheets of collagen fibres were presented as flattened tubular structures with oval entrances formed by the cut edges. The organisation of the middle zone into sheets was most clearly seen in para-
SEM of rabbit
incisor
median sections. The compartment walls clearly lay oblique to the cut surface and thus their edges were displayed lying parallel to the long axis of the tooth (Plate Fig. 9). The cut edges were almost always rolled over by the razor blade because the collagen was not supported by connection with the hard tissues as it was in transverse or median sections. At the corner of such specimens, there was a junction between the transverse and paramedian section planes. Stereo-pair photographs of these corners (Plate Fig. 10) confirmed that the bundles seen in transverse section, were in reality the cut edges of an overlapping interconnected array of sheets. Following treatment with a-amylase, the middle zone in transverse section (Plate Fig. 11) appeared to consist of a series of radially-flattened compartments which often contained ovoid bodies. In median sections treated with a-amylase (Plate Fig. 12), the middle zone appeared as a continuum of fibres. Numerous shallow depressions, circular or oval in outline, were evident. Ovoid bodies 5-7pm in length surrounded by a reticular framework were often found lying in the depressions. The ovoid bodies were interpreted as cell nuclei and the reticular material as cytoplasmic remnants. At a suitable tilt angle, the fibres exhibited a periodic cross-banding at S&60 nm. This detail was not visible in untreated specimens. DISCUSSION It was possible to observe with both light microscope and SEM three distinct zones in the rabbit periodontal ligament, represented diagrammatically in Text Fig. 13. The controversy about the existence of an intermediate plexus appears to have arisen as a result of the three-dimensional arrangement of fibres in the middle zone not having been recognized by two-dimensional histological techniques. Hindle (1967) found that the appearance of a plexus in the middle zone was much more evident in median section than in transverse section; this is so in the rabbit incisor (Plate Figs. 2 and 3). Thus, the appearance of an intermediate plexus arises because, in median section, the sheets of collagen lie in the section plane and are not cut transversely, and consequently do not resemble bundles, as they do in transverse section (Text Fig. 13). The function of the middle zone may be to allow the periodontal ligament both to provide support for the tooth and to allow the tooth to erupt. Sicher (1942) suggested that, in the intermediate plexus, the collagen fibres are continuously broken down and re-formed, thus allowing the tooth to move relative to the bone. Beertsen (1975) found that the cement and middle zones of the ligament move with the erupting tooth, whereas the alveolar fibres do not. He defined the plane of tissue shear as the interface between the middle and alveolar zones. His results do not include the possibility that the middle zone as a whole provides a plane of movement. In the rabbit incisor, the distinctive structure of the middle zone reported here may be very important in tissue remodelling. Breakdown of collagen during remodelling of the ligament may be accomplished by intracellular degradation of fibres within the fibroblasts (Ten Cate, 1972; Garant, 1976). The flattened shape of the fibroblasts within the middle
periodontium
6
Transverse
569
+
Fig. 13. Diagrammatic representation of the arrangement of the collagen fibres in the periodontal ligament, close to the alveolar crest. The fibres in the cement and alveolar zones are organised into bundles, whereas those in the arc arranged middle zone into sheets enclosing compartments.
zone would provide increased surface area for phagocytosis of fibres. Moreover, the sheets of collagen separating adjacent fibroblasts are only 0.5-l pm thick so that in the middle zone all regions of the collagen are easily accessible to the fibroblasts. This arrangement would allow rapid remodelling. In conformity with the absence of fibre bundles in the middle zone. all published electron micrographs seem to show that collagen in the process of intracellular degradation always consists of unit fibres. Piecemeal remodelling in this fashion, combined with the subdivision of the collagen into numerous. very thin sheets so that support could be maintained while fibre reorganisation was taking place, may be adaptations of the middle zone as a plane of shear. The middle zone may have mechanical properties different from the alveolar and cemental zones as, unlike them, it does not contain bundles. Biochemical studies of the periodontal ligament indicate abundant ground substances (Melcher and Walker, 1976) at least some of which are closely associated with the collagen (Pearson et al., 1975). In my study, the fine structure of collagen was visible only after incubation with a-amylase which removes ground substance (Hunter and Finlay, 1973) and then the middle zone appeared as a network of individual collagen fibres. These fibres are bound together by ground substance to form highly-ordered sheets. Minns, Soden and Jackson (1973), comparing a variety of connective tissues, suggest that in tendons the ground substances increase the strength of the tissue and confer visco-
510
P. Sloan
elastic properties. The arrangement of the middle zone into sheets might mean that stresses acting on one part of the ligament would be rapidly dispersed laterally and longitudinally. They also suggest that the collagen fibre orientation within a connective tissue may influence its mechanical properties. This may account for the highly-ordered nature of the fibres
Hunt
within the sheets and the variation in the arrangement of the sheets in periodontal ligament along the length of the tooth.
Eruption and Occlusion of Teeth (Edited by Poole D. F. G. and Stack M. V.) pp. 183-192. Butterworths. London. Minns R. J., Soden P. D. and Jackson D. S. 1973. The role of the fibrous components and ground substance in the mechanical properties of biological tissues: a preliminary investigation. J. Biomech. 6, 153-165. Ness A. R. and Smale D. E. 1959. The distribution 01 mitoses and cells in the tissue bounded by the socket wall of the rabbit mandibular incisor. Proc. R. SW. B. 151, 106128 Pearson C. H., Wohllebe M., Carmichael D. J. and ChoveIon A. 1975. Bovine periodontal ligament. An investigation of the collagen, glycosaminoglycan and insoluble glycoprotein components at different stages of tissue development. Conn. Tiss. Res. 3, 195-206. Sicher H. 1923. Bau und Funktion des Fixationsapparates der Meerschweinchenmolaren. Z. Sromat. 21, 58%594. Sicher H. 1942. Tooth eruption: the axial movement of continuously growing teeth. J. dent. Res. 21, 201-210. 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 okI Biol. 21, 633-635. Smith C. E. and Warshawskv H. 1976. Movement of entire cell populations during renewal of the rat incisor as shown by radioautography after labelling with 3Hthymidine. Am. J. Anat. 145, 225-260. Ten Cate A. R. 1972. Morphological studies of fibroblasts in connective tissue undergoing rapid remodelling. J. Amt. 112, 401-414.
Beertsen W. 1975. Migration of fibroblasts in the mouse periodontal ligament as revealed by autoradiography. Archs oral Biol. 20, 659-666. Beertsen W., Everts V. and van den Hooff A. 1974. Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. Archs orul Biol. 10, 1087-1098. Ciancio S. C., Neiders M. E. and Hazen S. P. 1967. The principal fibres of the periodontal ligament. Periodontics 5, 76-81. Garant R. P. 1976. Collagen resorption by fibroblasts. A theory of fibroblastic maintenance of the periodontal ligament. J. Periodont. 47, 38@390. Hindle M. 0. 1967. The intermediate plexus of the periodontal membrane. In: The Mechanisms of Tooth Support (Edited by Anderson D. J., Eastoe J. E., Melcher A. H. and Picton D. C. A.) pp.6671. John Wright, Bristol.
Plate transverse
Fig. 3. Rabbit
incisor:
median
Fig. 4. SEM
appearance
Fig. 5. SEM appearance
section section
close to the alveolar
of the ligament,
Fig. 6. Alveolar
zone, median
section.
Fig. 7. Cement
zone,
section.
median
A, Alveolar
zone;
transverse x200 median
Bundles
Fig. 8. Middle
section
Figs. 10 (A and B). Stereo-pair Fig.
section;
section.
of the junction section
11. Middle zone. Transverse
Fig. 12. Middle
zone. Median
in a region
with Sharpey
zone; C, cement
into
x 200 x 200
trichrome.
corresponding
corresponding
to Fig.
I.
to Fig. 2. x 200
fibres fan out into the middle
bundles
before
zone; BV, blood
to compartments of the sheets
between transversely face (right). x loo0 section section
azan trichrome. azan
being
inserted
into
vessel.
2.
entrances
The edges
Heidenhain
in a region
Collagen fibres group cementum. x 5000
M, middle
zone, oblique
zone, paramedian
crest. Heidenhain crest.
section
continuous zone. x 500
Plate Fig. 9. Middle
I.
close to the alveolar
of the ligament;
teeth
and
Int. Rev. corm. Tiss. Res. 6, 217-225.
REFERENCES
incisor:
of the molar
Melcher A. H. and Walker T. W. 1976. The periodontal ligament in attachment and as a shock absorber. In:
Acknowledgements-I wish to thank Mr. M. S. Gillett for his assistance with electron microscopy, Mr. J. E. Linder of the London Hospital Medical College who kindly stained many of the sections, and Dr. R. P. Shellis for his guidance in the preparation of the manuscript.
Fig. 2. Rabbit
A. M. 1959. A description
investing tissues of normal guinea pigs. J. dent. Res. 38, 216243. Hunter J. A. A. and Finlay B. 1973. Scanning electron microscopy of connective tissues in health and disease.
treated treated
appear
are rolled sectioned
x 100 x loo0
facet (left) and paramedian
with a-amylase. with a-amylase.
oval.
over in cutting.
x 2000 x loo0
SEM
of rabbit
incisor
Plate
periodonrium
I
571
P. Sloan
Plate 2
Dental
Unit,
SLOAN
The Dental School, Lower Maudlin Street. Bristol BSI 2LY. England
Summary-The structure of the rabbit incisor periodontium, prepared by slicing demineralised formalin-fixed jaws in various planes, was investigated. Three zones, corresponding to those recognised histologically, could be distinguished. The alveolar zone contained collagenous fibre bundles It%20 pm in diameter and large blood vessels. The collagen fibres in the middle zone were arranged in sheets which formed the structural basis of a series of radially-flattened compartments in the long axis of the tooth. Fibroblasts were often observed lying in the compartments. The collagen fibres in the cement zone were organised as bundles 3-5 pm in diameter. These appearances could be correlated with histological appearances; the arrangement of the fibres in the middle zone is in accord with current concepts of ligament remodelling.
lNTRODUCTlON
The arrangement of the collagen fibres in the periodontal ligament of continuously-erupting teeth has been the subject of controversy. On the basis of light microscope observations, Sicher (1923, 1942) described alveolar, middle and cement zones in the rat incisor and guinea-pig molar periodontal ligament, best seen in longitudinal sections. In the alveolar and cement zones, the collagen was organised as bundles which were continuous with the Sharpey fibres. The middle zone was composed of pre-collagenous fibres. Collagen fibres from the cement and alveolar zones could be traced into this zone for a short distance, forming the so-called intermediate plexus. An arrangement of the periodontal ligament into three zones was described in guinea-pig molar by Hunt (1959) and in rabbit incisor by Ness and Smale (1959). Using 0.5~pm sections, Smith and Warshawsky (1976) described an intermediate plexus in the periodontal ligament of the rat incisor. Beertsen, Everts and van den Hooff (1974) described variations in cell morphology and ultrastructure within the three zones in the rat incisor. Hindle (1967) traced fibre bundles across the periodontal ligament in the rat incisor in transverse sections but not in longitudinal sections, where the bundles were not continuous across the middle zone. Using polarizing microscopy, however, he found a reduction in the birefringence of the middle zone compared to the cement and alveolar zones in both transverse and longitudinal sections and thus favoured the concept of a middle zone composed mainly of pre-collagenous fibres. The existence of the intermediate plexus was disputed by Ciancio, Neiders and Hazen (1967) who traced fibre bundles across the periodontal ligament and considered that the appearance of an intermediate plexus was due to oblique sectioning. Sloan, Shellis and Berkovitz (1976) described effects of certain methods of specimen preparation on the appearance of rat periodontal ligament in the scanning electron microscope (SEM). Two techniques preserved the structure of the ligament; these were freeze-
fracture and slicing demineralized tissue with a razor blade, followed by critical-point drying. Because critical-point drying has the advantage that the plane of section can be controlled, it was decided to employ this method to examine the fibrous architecture of the ligament. The lower incisor of the rabbit was chosen because its size relative to the rat, for instance, permitted more accurate definition of the angulation and location of the section plane. MATERIALS
AND
METHODS
Seven young adult albino rabbits were killed by decapitation following anaesthesia with Nembutal. The mandibles were dissected out, divided in the rnidline and placed in 10 per cent neutral formal-saline. After two hours, the portions of the alveolar process containing the incisor teeth were separated from the rest of the jaw with a diamond disc and fixed for 7 days in fresh neutral formol-saline. The specimens were demineralised in 10 per cent ethylene diamine tetra-acetic acid (EDTA), pH 7.4, with continuous stirring. The solution was renewed weekly and the process continued until no mineral was detectable radiographically. After washing in 0.2 M phosphate buffer at pH 7.4 for 1 h, the incisors were serially divided into 3-mm slices with cuts transverse or oblique to the long axis of the tooth. Each slice was then divided in the median or paramedian plane (Text Fig. 1). After sectioning, all the blocks were stored in 0.2 M phosphate buffer. Some blocks which had been sliced transversely or in median planes were selected and incubated in crude bacterial r-amylase (BDH Chemicals Ltd., Poole, Dorset) at a concentration of I mg/ml in 0.2 M phosphate buffer at 37°C for 2 h to remove ground substance (Hunter and Finlay, 1973). All specimens were then washed in distilled water, dehydrated in a graded ethanol series, critical-point dried in amyl acetate from carbon dioxide, mounted on Stereoscan stubs and coated with carbon and gold/ palladium alloy. The specimens were examined in a Stereoscan 600 (Cambridge Instruments Ltd., Cam567
568
P. Sloan
Fig. 1. Section
planes
used in the SEM study.
bridge, England) at an accelerating voltage of 15 V, using a variety of tilt angles. Stereo-pair photographs of the ligament in each section plane and of the regions where two planes intersected were taken. Two pairs of demineralised lower incisors were dehydrated and embedded in paraffin wax. Serial sections were cut at 8 m in the transverse and median planes and stained with Ehrlich haematoxylineosin or Heidenhain azan trichrome. RESULTS
Histological observations Only findings which are relevant to understanding SEM appearances will be described here. In both the transverse and median planes, three zones within the periodontal ligament could be distinguished (Plate Figs. 2 and 3). The alveolar zone occupied approximately 40 per cent of the ligament width and contained the main periodontal blood vessels, which were larger and more numerous at the basal end of the tooth. The collagen fibres in the alveolar zone were organised as large bundles which coursed between the blood vessels and were inserted into the bone at approximately right angles to the mineralized surface. The fibroblasts in this region were spindle-shaped in both transverse and median sections. The middle zone occupied about 50 per cent of the width of the ligament. In transverse section, it appeared to consist of radially-orientated bundles which arose by subdivision of the larger bundles in the alveolar zone. The bundles in the middle zone branched and anastomosed with each other (Plate Fig. 2). In median section, however, the fibres of the middle zone did not appear to be organised as large bundles, but appeared as a meshwork of individual fine fibres (Plate Fig. 3). The fibroblasts in the middle zone were spindle-shaped in transverse section, but in median section had approximately circular outlines.
Median
refers to the midline
of the tooth
The cement zone occupied up to 10 per cent of the ligament width and was similar in transverse and median sections. The collagen fibres were organised as circular bundles somewhat smaller than those in the alveolar zone, which were separated by cuboidal cells. SEA4 observations In transverse sections (Plate Fig. 4) alveolar, middle and cement zones could be distinguished. The alveolar zone occupied about 40 per cent of the width of the ligament and contained bundles lC20pm in diameter, composed of collagen fibres. These were continuous with the Sharpey fibres. The middle zone appeared to consist of fibre bundles l-3 pm in diameter which were separated by a distance of 0.5-I pm and arose by branching of the larger bundles in the alveolar zone. They formed a continuous network by branching and anastomosing with each other. Branching occurred at 3%10pm intervals. In places, the bundles were wavy in the transverse plane and were always radially orientated on the medial aspect of the tooth. The orientation of the fibres in the lateral parts of the ligament changed along the length of the tooth; in the alveolar crest region, they passed straight from cement to bone but followed a progressively more oblique course towards the growing end. The bundles in the cement zone were 3-5 pm in diameter and were orientated at approximately right angles to the mineralized surface. In the median plane, the collagen in the alveolar and cement zones was also organised as bundles (Plate Fig. 5) which fanned out into the middle zone (Plate Figs. 6 and 7). In this plane, the middle zone appeared as an undulating, sheet-like continuum of fibres which formed a series of compartments. In oblique sections (Plate Fig. 8), the compartments formed by the sheets of collagen fibres were presented as flattened tubular structures with oval entrances formed by the cut edges. The organisation of the middle zone into sheets was most clearly seen in para-
SEM of rabbit
incisor
median sections. The compartment walls clearly lay oblique to the cut surface and thus their edges were displayed lying parallel to the long axis of the tooth (Plate Fig. 9). The cut edges were almost always rolled over by the razor blade because the collagen was not supported by connection with the hard tissues as it was in transverse or median sections. At the corner of such specimens, there was a junction between the transverse and paramedian section planes. Stereo-pair photographs of these corners (Plate Fig. 10) confirmed that the bundles seen in transverse section, were in reality the cut edges of an overlapping interconnected array of sheets. Following treatment with a-amylase, the middle zone in transverse section (Plate Fig. 11) appeared to consist of a series of radially-flattened compartments which often contained ovoid bodies. In median sections treated with a-amylase (Plate Fig. 12), the middle zone appeared as a continuum of fibres. Numerous shallow depressions, circular or oval in outline, were evident. Ovoid bodies 5-7pm in length surrounded by a reticular framework were often found lying in the depressions. The ovoid bodies were interpreted as cell nuclei and the reticular material as cytoplasmic remnants. At a suitable tilt angle, the fibres exhibited a periodic cross-banding at S&60 nm. This detail was not visible in untreated specimens. DISCUSSION It was possible to observe with both light microscope and SEM three distinct zones in the rabbit periodontal ligament, represented diagrammatically in Text Fig. 13. The controversy about the existence of an intermediate plexus appears to have arisen as a result of the three-dimensional arrangement of fibres in the middle zone not having been recognized by two-dimensional histological techniques. Hindle (1967) found that the appearance of a plexus in the middle zone was much more evident in median section than in transverse section; this is so in the rabbit incisor (Plate Figs. 2 and 3). Thus, the appearance of an intermediate plexus arises because, in median section, the sheets of collagen lie in the section plane and are not cut transversely, and consequently do not resemble bundles, as they do in transverse section (Text Fig. 13). The function of the middle zone may be to allow the periodontal ligament both to provide support for the tooth and to allow the tooth to erupt. Sicher (1942) suggested that, in the intermediate plexus, the collagen fibres are continuously broken down and re-formed, thus allowing the tooth to move relative to the bone. Beertsen (1975) found that the cement and middle zones of the ligament move with the erupting tooth, whereas the alveolar fibres do not. He defined the plane of tissue shear as the interface between the middle and alveolar zones. His results do not include the possibility that the middle zone as a whole provides a plane of movement. In the rabbit incisor, the distinctive structure of the middle zone reported here may be very important in tissue remodelling. Breakdown of collagen during remodelling of the ligament may be accomplished by intracellular degradation of fibres within the fibroblasts (Ten Cate, 1972; Garant, 1976). The flattened shape of the fibroblasts within the middle
periodontium
6
Transverse
569
+
Fig. 13. Diagrammatic representation of the arrangement of the collagen fibres in the periodontal ligament, close to the alveolar crest. The fibres in the cement and alveolar zones are organised into bundles, whereas those in the arc arranged middle zone into sheets enclosing compartments.
zone would provide increased surface area for phagocytosis of fibres. Moreover, the sheets of collagen separating adjacent fibroblasts are only 0.5-l pm thick so that in the middle zone all regions of the collagen are easily accessible to the fibroblasts. This arrangement would allow rapid remodelling. In conformity with the absence of fibre bundles in the middle zone. all published electron micrographs seem to show that collagen in the process of intracellular degradation always consists of unit fibres. Piecemeal remodelling in this fashion, combined with the subdivision of the collagen into numerous. very thin sheets so that support could be maintained while fibre reorganisation was taking place, may be adaptations of the middle zone as a plane of shear. The middle zone may have mechanical properties different from the alveolar and cemental zones as, unlike them, it does not contain bundles. Biochemical studies of the periodontal ligament indicate abundant ground substances (Melcher and Walker, 1976) at least some of which are closely associated with the collagen (Pearson et al., 1975). In my study, the fine structure of collagen was visible only after incubation with a-amylase which removes ground substance (Hunter and Finlay, 1973) and then the middle zone appeared as a network of individual collagen fibres. These fibres are bound together by ground substance to form highly-ordered sheets. Minns, Soden and Jackson (1973), comparing a variety of connective tissues, suggest that in tendons the ground substances increase the strength of the tissue and confer visco-
510
P. Sloan
elastic properties. The arrangement of the middle zone into sheets might mean that stresses acting on one part of the ligament would be rapidly dispersed laterally and longitudinally. They also suggest that the collagen fibre orientation within a connective tissue may influence its mechanical properties. This may account for the highly-ordered nature of the fibres
Hunt
within the sheets and the variation in the arrangement of the sheets in periodontal ligament along the length of the tooth.
Eruption and Occlusion of Teeth (Edited by Poole D. F. G. and Stack M. V.) pp. 183-192. Butterworths. London. Minns R. J., Soden P. D. and Jackson D. S. 1973. The role of the fibrous components and ground substance in the mechanical properties of biological tissues: a preliminary investigation. J. Biomech. 6, 153-165. Ness A. R. and Smale D. E. 1959. The distribution 01 mitoses and cells in the tissue bounded by the socket wall of the rabbit mandibular incisor. Proc. R. SW. B. 151, 106128 Pearson C. H., Wohllebe M., Carmichael D. J. and ChoveIon A. 1975. Bovine periodontal ligament. An investigation of the collagen, glycosaminoglycan and insoluble glycoprotein components at different stages of tissue development. Conn. Tiss. Res. 3, 195-206. Sicher H. 1923. Bau und Funktion des Fixationsapparates der Meerschweinchenmolaren. Z. Sromat. 21, 58%594. Sicher H. 1942. Tooth eruption: the axial movement of continuously growing teeth. J. dent. Res. 21, 201-210. 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 okI Biol. 21, 633-635. Smith C. E. and Warshawskv H. 1976. Movement of entire cell populations during renewal of the rat incisor as shown by radioautography after labelling with 3Hthymidine. Am. J. Anat. 145, 225-260. Ten Cate A. R. 1972. Morphological studies of fibroblasts in connective tissue undergoing rapid remodelling. J. Amt. 112, 401-414.
Beertsen W. 1975. Migration of fibroblasts in the mouse periodontal ligament as revealed by autoradiography. Archs oral Biol. 20, 659-666. Beertsen W., Everts V. and van den Hooff A. 1974. Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. Archs orul Biol. 10, 1087-1098. Ciancio S. C., Neiders M. E. and Hazen S. P. 1967. The principal fibres of the periodontal ligament. Periodontics 5, 76-81. Garant R. P. 1976. Collagen resorption by fibroblasts. A theory of fibroblastic maintenance of the periodontal ligament. J. Periodont. 47, 38@390. Hindle M. 0. 1967. The intermediate plexus of the periodontal membrane. In: The Mechanisms of Tooth Support (Edited by Anderson D. J., Eastoe J. E., Melcher A. H. and Picton D. C. A.) pp.6671. John Wright, Bristol.
Plate transverse
Fig. 3. Rabbit
incisor:
median
Fig. 4. SEM
appearance
Fig. 5. SEM appearance
section section
close to the alveolar
of the ligament,
Fig. 6. Alveolar
zone, median
section.
Fig. 7. Cement
zone,
section.
median
A, Alveolar
zone;
transverse x200 median
Bundles
Fig. 8. Middle
section
Figs. 10 (A and B). Stereo-pair Fig.
section;
section.
of the junction section
11. Middle zone. Transverse
Fig. 12. Middle
zone. Median
in a region
with Sharpey
zone; C, cement
into
x 200 x 200
trichrome.
corresponding
corresponding
to Fig.
I.
to Fig. 2. x 200
fibres fan out into the middle
bundles
before
zone; BV, blood
to compartments of the sheets
between transversely face (right). x loo0 section section
azan trichrome. azan
being
inserted
into
vessel.
2.
entrances
The edges
Heidenhain
in a region
Collagen fibres group cementum. x 5000
M, middle
zone, oblique
zone, paramedian
crest. Heidenhain crest.
section
continuous zone. x 500
Plate Fig. 9. Middle
I.
close to the alveolar
of the ligament;
teeth
and
Int. Rev. corm. Tiss. Res. 6, 217-225.
REFERENCES
incisor:
of the molar
Melcher A. H. and Walker T. W. 1976. The periodontal ligament in attachment and as a shock absorber. In:
Acknowledgements-I wish to thank Mr. M. S. Gillett for his assistance with electron microscopy, Mr. J. E. Linder of the London Hospital Medical College who kindly stained many of the sections, and Dr. R. P. Shellis for his guidance in the preparation of the manuscript.
Fig. 2. Rabbit
A. M. 1959. A description
investing tissues of normal guinea pigs. J. dent. Res. 38, 216243. Hunter J. A. A. and Finlay B. 1973. Scanning electron microscopy of connective tissues in health and disease.
treated treated
appear
are rolled sectioned
x 100 x loo0
facet (left) and paramedian
with a-amylase. with a-amylase.
oval.
over in cutting.
x 2000 x loo0
SEM
of rabbit
incisor
Plate
periodonrium
I
571
P. Sloan
Plate 2
