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Differential distribution and ultrastructural staining of oxytalan and elastic fibers in the periodontal ligament of Alligator mississippiensis.

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THE ANATOMICAL RECORD 225:279-287 (1989)
Differential Distribution and Ultrastructural
Staining of Oxytalan and Elastic Fibers in the
Periodontal Ligament of Alligator mississippiensis
MINORU TAKAGI, TOSHITADA KAZAMA, KAZUYUKI SHIMADA,
YASUNOBU HOSOKAWA, AND HIDEKI HISHIKAWA
Departments of Anatomy (M.T., K.S., Y.H., H.H.) and Oral Surgery (T.K.),
Nihon University School of Dentistry, Tokyo 101, Japan
ABSTRACT
We have investigated ultrastructural cytochemical properties of
elastic elements in Alligator periodontal ligaments decalcified with EDTA and
stained with 1)the tannic acid-uranyl acetate (TA-UA) method for elastin in combination with elastase digestion; 2) the high iron diamine-thiocarbohydrazidesilver proteinate (HID-TCH-SP) method with prior treatment of specimens with
either monopersulphate or cupric-sulphite reagent for the localization of disulphide- and/or sulphydryl-containing material (i.e., oxytalan fibers); and 3) HIDTCH-SP alone for sulphated complex carbohydrates. Many microfibrils accumulated t o form either large or small bundles. Large bundles having a diameter of
2.50 1.10 pm (mean & SD; n-50) each showed an apico-occlusal distribution,
although small bundles measuring 0.63 0.13 km (mean k SD; n = 50) in diameter each were exclusively localized in interstitial areas rich in vessels and nerves.
The former bundles always lacked TA-UA reactivity and represented oxytalan
fibers; the latter bundles frequently contained TA-UA-reactive elastase digestable
components and were similar in appearance to immature elastic fibers or elaunin
fibers. HID-TCH-SP after oxidation strongly stained both the oxytalan and elastic
fiber microfibrils but stained the amorphous elastin very weakly or not all. In
nonoxidized specimens, there was no definite HID-TCH-SP staining of microfibrils
and the amorphous elastin, although adjacent matrix proteoglycans stained consistently. These results indicate that although there is a marked difference in the
distribution and size of oxytalan and elastic fibers in Alligator periodontal ligaments, their associated microfibrils lack stainable sulphate groups but are enriched with disulphide andlor sulphydryl groups, as has been described in mammals.
*
The periodontal ligament is the connective tissue
that surrounds the root of the tooth and attaches it to
the alveolar bone. The stresses of biting are taken up
by connective tissue fibers that compose the bulk of the
ligament. The predominant fibers localized here are
undoubtedly collagen fibers (Fullmer et al., 1974). Oxytalan fibers, another fibrous element, can be identified
in mammals such as humans, monkeys, rats, mice,
and guinea pigs by elastic stains such as Gomori’s aldehyde fuchsin and orcein following peracetic acid
or Oxone@ (monopersulphate compound) (2KHS05.
KHS04.K2S04,Du Pont Co.) oxidation, whereas typical elastic fibers with a distribution unrelated to blood
and lymphatic vessels are rarely seen in these animals
(Fullmer, 1958, 1960; Fullmer and Lillie, 1958;
Fullmer et al., 1974). However, distinct elastic fibers
whose distribution is similar to that of oxytalan fibers
are thought t o be present in some other mammals
(Fullmer et al., 1974). An intermediate fiber between
oxytalan and elastic fiber, termed elaunin fiber, stains
like oxytalan fibers, except that orcein stains without
oxidation (Gawlik, 1965).This fiber type has been dem0 1989 ALAN R. LISS, INC.
*
onstrated in human skin at the light and electron microscopic level (Gawlik, 1965; Cotta-Pereira et al.,
1976) but not in the periodontal ligament.
The emergence of socketed teeth attached to the alveolar bone with the periodontal ligament can only be
seen in reptiles amongst the nonmammalian vertebrates. The only living reptiles with this type of attachment are the Crocodilia such as Crocodile, Alligator,
and Caiman (Shellis, 1981). Light microscopic studies
(Soule, 1967) have localized both oxytalan fibers and
elastic fibers in the periodontal ligament of the Alligator and the Caiman. However, ultrastructural studies
required t o distinguish these fibers have not been conducted.
Recently, oxytalan fibers have been stained and visualized by light and electron microscopy (Hirayama et
Received January 17, 1989; accepted March 20, 1989.
Address reprints request to Minoru Takagi, D.D.S., Ph.D., Department of Anatomy, Nihon University School of Dentistry, 1-8-13
Kanda-Surugadai, Chiyoda-ku, Tokyo 101, Japan.
280
M. TAKA(;I ET AL.
al., 1985; Takagi et al., 1987) using Spicer’s high iron
diamine (HID) method (Spicer, 1965; Spicer et al.,
1967) and the HID-thiocarbohydrazide-silverproteinate (TCH-SP) method (Sannes et al., 1979) after oxidation of monkey periodontal ligaments with either Oxone or cupric-sulphite reagent. The specificity of the
HID method for sulphate groups has been extensively
described previously (Lev and Spicer, 1965; Spicer,
1965; Spicer e t al., 1967; Gad and Sylven, 1969; Sorvari, 1972). The HID-reactive sites can be further enhanced by treatment with TCH-SP (Sannes et al.,
1979). Peracetic acid or Oxone is thought to oxidize
disulphide linkages to sulphonic acid groups (Fullmer
et al., 1974; Hirayama et al., 1985) and possibly sulphuric acid; the cupric sulphite treatment oxidizes disulphides and sulphydryls to cysteine S-sulphonic acid
(Castino and Bussolati, 1974). These oxidative products may take part in the HID and HID-TCH-SP staining reaction (Takagi et al., 1988; Baba et al., 1988). The
present study of Alligator periodontal ligaments utilized these stains together with the tannic acid-uranyl
acetate (TA-UA) method for elastin (Kageyama et al.,
1985) to distinguish various fiber types and provide
comparison to mammalian species.
MATERIALS AND METHODS
An Alligator mississippiensis with a n overall length
of 125 cm was sacrificed. The mandible and the maxilla
were fixed in neutral formalin for 3 months and then
demineralized for about 6 months at 4°C in 10% EDTA
in 0.1 M cacodylate buffer, pH 7.3, with constant agitation and then rinsed in 0.1 M cacodylate buffer (pH
7.3). The demineralized specimens were cut into small
blocks, each containing teeth with their surrounding
periodontal tissues. For the light microscopic examination, specimens were dehydrated and embedded in celloidin. To demonstrate oxytalan fibers, sections (20 pm
thick) were stained with orcein solution (Unna, 1894)
for 30 min or Spicer’s (1965; Spicer et al., 1967) HID
solution 6-18 h r after treatment with either Oxone for
30 and 60 min or a cupric-sulphite reagent for 3 h r as
described previously (Takagi et al., 1987; Baba et al.,
1988). Control sections were also stained with the orcein or the HID solution without oxidation. Orcein solution was prepared by adding l g orcein (E. Merck,
Darmstadt, West Germany) and 1ml concentrated HC1
in 100 ml of 70% aqueous ethanol (Unna, 1894). Preparation of the HID stain and oxidative solution is described below. Specimens for ultrastructural studies
were further reduced in size by slicing with a razor
blade and refixed in Karnovsky’s (1965) fixative (modified) containing 4% paraformaldehyde-0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 h r a t
4°C. Subsequently, the specimens were thoroughly
rinsed in 0.1 M cacodylate buffer (ph 7.3) and were
processed a s outlined below.
Morphology
Some specimens were postfixed for 1h r in 1%Os04
in 0.1 M cacodylate buffer pH 7.3, then dehydrated in
graded ethanols and propylene oxide, and embedded in
Spurr’s (1969) low-viscosity resin. Thin sections (70-75
nm thick) were cut with a diamond knife using a Sorvall MT-5000 ultramicrotome and counterstained with
uranyl acetate and Sato’s (1968) triple lead. All speci-
mens were examined in a Hitachi H-500 electron microscope at a n accelerating voltage of 75 kV. The size of
fiber bundles, microfibrils, and reaction products was
measured on prints, using a hand magnifier equipped
with a 0.1 mm scale. Mean measurements were given
with their standard deviation and the number of samples measured.
Tannic Acid-Uranyl Acetate (TA-UA) Method for Elastin
Thin sections were mounted on stainless steel grids
and stained with the TA-UA method, as previously described (Kageyama et al., 1985). They were treated for
10 min with a filtered fresh TA solution (pH 7.0) containing 5 g TA (gallotannic acid; J.T. Baker Chemical
Co., Phillipsburg, NJ) in 95 ml distilled water. The pH
of the TA solution was adjusted with 10 N NaOH. After
TA treatment, thin sections were then rinsed by dipping into three successive baths of distilled water for 1
min and immediately treated for 5 min with a filtered
fresh UA solution prepared by adding 1 g UA (uranyl
acetate; E. Merck, Darmstadt, West Germany) in 99 ml
of distilled water. Subsequently, treated thin sections
were rinsed in distilled water and examined. Some TAUA-treated thin sections were stained with Sato’s
(1968) triple lead for 1min. As a control, thin sections
without TA treatment were examined after exposure to
either the UA or the triple lead alone.
Enzymatic Digestion of Elastin
The aldehyde-fixed rinsed specimens were immersed
in 0.2 M Tris-HC1 buffer (pH 8.8) and then exposed for
1 h r a t 37°C to 2 ml solution containing 44 units of
elastase (from porcine pancreas, E.C. 3 . 4 . 21. l l . , type
IV, 88 units per mg: Sigma Chemical Co., St. Louis,
MO) in 0.2 M Tris-HC1 buffer (pH 8.8) and then rinsed
several times in 0.2 M Tris-HC1 buffer. Control specimens were incubated for 1 hr in 0.2 M Tris-HC1 buffer
(pH 8.8) without elastase a t 37°C and then rinsed several times in 0.2 M Tris-HC1 buffer. Subsequently, the
specimens were postosmicated, dehydrated, and embedded as described above. The thin sections were
mounted on stainless steel grids and stained with TAUA a s described above.
HID and HID-TCH-SP Methods With and Without
Monopersulphate Oxidation
The aldehyde-fixed, rinsed specimens, approximately 0.3 x 0.5 x 2.0 mm in size, were treated with
10% Oxone for 60 min and were thoroughly rinsed in
distilled water. The Oxone was obtained from Aldrich
Chemical Co., (Milwaukee, WI). Some specimens were
also processed without Oxone treatment. The specimens were then stained for 18 h r a t 22°C in the HID
solution (Spicer, 1965; Spicer et al., 1967) prepared by
adding 1.4 ml 40% FeC13 (Fisher Scientific Co., Fair
Lawn, NJ) to a fresh diamine solution containing 120
mg N,N-dimethyl-m-phenylenediaminedihydrochloride (Eastman Kodak Co., Rochester, NY) and 20 mg
N,N-dimethyl-p-phenylenediamine monohydrochloride (Fisher Scientific) in 50 ml distilled water. Control
specimens (for evaluation of intrinsic density) were incubated for 18 h r at 22°C in 1%MgC12, pH 1.4, adjusted
with HC1. Some specimens were then postfixed in 1%
OsO4 in 0.1 M cacodylate buffer (pH 7.3), dehydrated,
ALLIGATOR PERIODONTAL LIGAMENTS: ELASTIC ELEMENTS
281
Fig. 1. In the Alligator periodontal ligaments, Oxone-orcein
strongly stains fibers (thick arrows and enlarged in b) with a n apicoocclusal alignment and small punctuated material (thin arrows and
enlarged in b) in interstitial spaces. Some fibers (arrowhead) appear
to enter the cementum (C) but not the alveolar bone (B). White arrow,
blood capillaries. a, x 140; b, x 560.
and embedded in Spurr’s low-viscosity resin. The postosmication step was omitted for other specimens.
To enhance HID staining, some thin sections were
stained with a thiocarbohydrazide (Eastman Kodak)silver proteinate (strong silver protein, Roboz Surgical
Instrument Co., Washington, DC) sequence as described previously (Sannes et al., 1979). Silver background staining was elminiated by filtering (Whatman
filter No. 2) the silver proteinate solution twice before
use. Acid MgClz controls were similarly processed. All
specimens were examined without uranyl acetate and
lead citrate counterstaining.
RESULTS
Light Microscopy
Thiosulphation
A histochemical method for the demonstration of
protein-bound disulphide and sulphydryl groups after
thiosulphation was ultrastructurally employed, since
previous reports indicated that the induced thiosulphate groups share the histochemical reactions of organic ester sulphate groups (Castino and Bussolati,
1974). Specimens were brought to distilled water,
treated for 1 min in 0.5 M NH,OH, and then incubated
for 3 hr a t 22°C in a cupric-sulphite regent prepared
before use by mixing 40 m10.5 M ammonia solution, 40
mlO.025 M cupric sulphate brought to pH 9 by adding
a few drops of concentrated ammonia, and 10 m10.2 M
sodium metabisulphite (Sigma) as described previously
(Castino and Bussolati, 1974). Control specimens were
incubated for 3 hr a t 22°C in the reagent without sodium metabisulphite. Subsequently, all specimens
were rinsed in distilled water and stained with HID.
Some specimens were then postfixed in 1%OsO4 in 0.1
M cacodylate buffer (pH 7.3), dehydrated, and embedded. The postosmication step was omitted for other
specimens. Some thin sections were stained with TCHSP as described above.
In Alligator periodontal ligmants following prior
treatment with either Oxone (Fig. 1;Table 1)or cupricsulphite reagent (not illustrated; Table l), both orcein
and HID strongly stained fibers which ran parallel to
the long axis of the tooth, from the region near to the
apex of the root t o the cervical region near to the cemento-enamel junction. These longitudinal fibers were
always distributed closer to the tooth side of the periodontal ligament than on the alveolar bone side. Some
fibers branched toward the tooth and were often inserted into cementum but were not found near or into
the alveolar bone. Strong staining was also closely associated with small punctuated material in “the median zone,” a term previously suggested by Soule
(19671, characterized by nerves and numerous small
vascular vessels and presumed to represent interstitial
spaces in human periodontal ligaments. In unoxidized
specimens, fibers with an apico-occlusal alignment
lacked orcein staining, but the punctuated material
stained strongly (Table 1): HID staining was not seen
in these sites in unoxidized specimens (Table 1).
Electron Microscopy
Morphology
There were more microfibrils observed on the tooth
side of the Alligator periodontal ligament than on the
alveolar bone side. These microfibrils appeared to accumulate and to form either large or small bundles.
Large bundles, each bundle having a diameter of 2.50
-t 1.10 pm (mean -t SD; n=50), were interspersed
among collagen fibers and always lacked amorphous
elastin-like material (Fig. 2), whereas smaller bundles
having a diameter of 0.63 2 0.13 pm (mean k SD;
n = 50) were exclusively localized in the median zone o r
282
M. TAKAGI ET AL.
TABLE 1A. Light microscopic histochemistry of
Alligator periodontal ligaments’
Fibers with an
apico-occlusal
alignment’
Punctuated
material in
the median zone3
+
+
+
+
+
+
-
-
Oxone-orcein
Thiosulphation-orcein
Orcein
Oxone-HID
Thiosulphation-HID
+
-
+
HTT)
+
TABLE 1B. Ultrastructural staining of AZZigutor
periodontal ligaments
MorDholoev
TA-UA
Elastase-TA-UA
Oxone-HID-TCH-SP
HID-TCH-Sp
ThiosulphationHID-TCH-SP
Large bundle’
Small bundle3
Microfibril Microfibril Amorphous
bundles
bundles
material
-
-
-
-
+
-5
+
+
-5
+
+
-
-
‘Staining reaction: + , positive; -, negative
‘Fibers corresponded to large bundles that were ultrastructurally
identified as oxytalan fibers.
3The punctuated material corresponded to small bundles with and
without amorphous material that were ultrastructurally identified as
immature elastic fibers or elaunin.
4HID-TCH-SPstaining was evaluated in unosmicated specimens.
‘Microfibrils did not demonstrate definite HID-TCH-SP staining but
contained few stain deposits. However, it is not known whether this
staining represents “true” or “background staining.
HID-TCH-SP method with and without monopersulphate
oxidation
HID-TCH-SP treatment following monopersulphate
oxidation strongly stained both the microfibril bundles,
but definitive staining was not seen in amorphous elastin (Figs. 7, 8). The stain deposits having larger size (a
diameter of 6-22 nm) were exclusively localized in the
microfibrils, whereas reaction deposits having smaller
size (a diameter of 4-7 nm) persisted in collagen associated-matrix material, presumed to be proteoglycans
(Figs. 7, 8).
The HID-TCH-SP method without monopersulphate
oxidation weakly stained matrix material in close association collagen fibrils, whereas staining was not apparent in both the microfibrils and amorphous elastin
(Figs. 9, 10). However a few HID-TCH-SP stain deposits were localized in these sites. This latter staining
could not be clearly distinguished from ‘background’
staining.
As controls for the HID-TCH-SP procedure, TCH-SP
treatment of specimens which were processed with and
without oxidation and incubated in an acid MgClz control solution lacked staining in the aforementioned
sites.
Thiosulphation
Treatment with cupric-sulphite reagent similarly
strongly enhanced subsequent HID-TCH-SP staining
of both the microfibril bundles and only weakly stained
matrix material surrounding collagen fibrils, whereas
significant HID-TCH-SP stain deposits were not observed in the amorphous elastin (Figs. 11-13).
Control specimens incubated in reagent without sodium metabisulphite lacked significant HID-TCH-SP
staining of both the microfibrils, except for weak staining of the matrix material surrounding collagen fibrils.
DISCUSSION
interstitial spaces; and most, but not all of the small
The present study of the periodontal ligament in the
bundles contained centrally located amorphous material (Figs. 3, 4). Large bundles were rarely found in Alligator mississippiensis demonstrates differential
interstitial spaces. Thus, large and small bundles cor- distribution of oxytalan fibers having an apico-occlusal
responded to fibers and the punctuated material that alignment and immature elastic fibers or elaunin in
were identified in light microscopic studies. However, interstitial spaces rich in vessels and nerves at the
there were no significant differences of microfibrils light microscopic level, and further correlates both the
composing both of the bundles. The diameter of mi- fibers with two types (large and small) of microfibril
crofibrils localized in large and small bundles mea- accumulations at the ultrastructural level. Both the
sured 12.0 2.0 nm (mean k SD; n = 50) and 12.0 k 2.2 microfibril accumulations are morphologically distinct
nm (mean k SD; n = 50), respectively; both microfibrils and can be easily identified by their distribution and
had a light central core surrounded by a dense outer size, and also by the absence or presence of elastin.
layer and exhibited a beaded pattern along their long Individual microfibrils from both accumulations have a
similar morphology and stain positively for disulphide
axis, when observed in cross or longitudinal section.
*
TA-UA method and elastase digestion
TA-UA staining could not be observed in large bundles (not illustrated) or in small bundles devoid of
amorphous material (Fig. 5). However, TA-UA
strongly stained amorphous material in small microfibril bundles and moderately stained collagen
fibrils (Fig. 5). In elastase-digested specimens, all TAUA-reactive amorphous material was selectively removed (Fig. 6 ) , whereas buffer controls with TA-UA
treatment demonstrated strong staining of amorphous
material.
Fig. 2. a: Large bundles (thick arrow and enlarged in b) in the
Alligator periodontal ligaments contain abundant microfibrils but not
amorphous elastin-like material and are in close association with collagen fibrils (CO).Cross-sectioned and longitudinally sectioned microfibrils (thin arrows) are visible in this bundle. a, ~ 1 7 , 5 0 0 b,
;
x 45,000.
Figs. 3, 4. Numerous small microfibril bundles are localized in close
proximity to blood capillaries (white arrow) in the intersititial space;
most of them (arrows) contain centrally located elastin-like material
(E), whereas some (arrowheads) appear to lack this material. CO,collagen fibrils. Counterstained with uranyl acetate and triple lead. Figure 3, x 17,500. Figure 4, x 45,000.
ALLIGATOR PERIODONTAL LIGAMENTS: ELASTIC ELEMENTS
Figs. 2-4.
283
284
M. TAKAGI ET AL.
Fig. 5. TA-UA staining. In the interstitial space, TA-UA-reactive
amorphous elastin (E) can be visualized in the majority of small microfibril bundles (arrows, and enlarged in insets), but some (arrowhead, and enlarged in inset) lack this reactive material. C O ,collagen
fibrils. Specimen weakly counterstained with triple lead. x 17,500;
insets, x 45,000.
Fig. 6. TA-UA staining following en bloc elastase treatment. Enzyme digestion removes all TA-UA-reactive amorphous elastin in
small microfibril bundles (arrows, and enlarged in inset) in the interstitial space. CO,collagen fibrils. Specimen weakly counterstained
with triple lead. x 17,500; inset x 45,000.
and/or sulphydryl groups, but lack stainable sulphate
groups utilizing the HID-TCH-SP method. The present
histochemical results are consistent with previous
light microscopic studies (Soule, 1967) of the Alligator
periodontal ligament identifying oxytalan and elastic
fibers in these sites, utilizing aldehyde fuchsin with
and without oxidation with peracetic acid. The present
study is the first to extend these observations to the
ultrastructural level and correlates the morphology
and the staining characteristics with distinct oxytalan
and elastic fibers in the periodontal ligaments of the
Alligator.
Large microfibril bundles can be identified as oxytalan fibers by the lack of stainable elastin regardless of
the abundant microfibril accumulations. This observation is consistent with previous ultrastructural studies
(Carmichael and Fullmer, 1966; Fullmer et al., 1974)
demonstrating similar morphological observations of
Fig. 7. Oxone-oxidized specimen, HID-TCH-SP staining with postosmication. Intense staining is observed in large microfibril bundles
(arrow, and enlarged in inset), whereas the matrix material associated with collagen (CO,enlarged in inset) is stained weakly. Large
stain deposits are exclusively localized in their microfibrils whereas
smaller stain deposits are seen in the periphery of collagen fibrils.
Specimen not counterstained. x 17,500, inset, x 45,000.
Fig. 8. Oxone-oxidized specimen, HID-TCH-SP staining without
postosmication. In the interstitial space, large stain deposits can be
visualized in microfibrils in small bundles (arrow, and enlarged in
b), whereas amorphous material (E) lacks staining. Small stain deposits (arrowhead) are observed in the matrix material surround-
ing collagen fibrils. Specimen not counterstained. a, x 17,500;
b, ~ 4 5 , 0 0 0 .
Figs. 9, 10. Unoxidized specimen, HID-TCH-SP staining with postosmication. Stain deposits (arrowheads in Figs. 9 and 10a) can be seen
in the matrix material closely associated with collagen fibrils (CO,and
enlarged in inset of Fig. 9 and in Fig. lob). Large bundles or oxytalan
fibers (thick arrows in Fig. 9, and enlarged in inset) as well as small
bundles or immature elastic fibers (white arrow in Fig. 10a and enlarged in Fig. lob) do not demonstrate definite staining, although a
few stain deposits may be identified (thin arrows in Figs. 9 and lob).
Specimen not counterstained. Figures 9 and 10a, x 17,500; inset and
Figure lob, x 45,000.
286
M. TAKAGI ET AL.
Figs. 11-13, HID-TCH-SP staining following thiosulphation. Stain
deposits are found in microfibrils forming large bundles (thick arrow,
and enlarged in inset of Fig. 11)and small bundles (arrowheads in
Figs. 12 and 13) with or without amorphous elastin (E). The stain
deposits are more numerous and larger than those in the matrix material in association with collagen fibrils (CO).Amorphous elastin
lacks definitive staining. Unosmicated specimen not counterstained.
Figures 11 and 12, X 17,500; inset and Figure 13, x 45,000.
oxytalan fibers in mammaiian periodontal ligaments. the principal collagen fiber bundles may provide the
In contrast, small microfibril bundles are characterized periodontal ligament with special elasticity required
by the decreased microfibril accumulations with or for biting. On the other hand, elastic fibers, which dewithout the presence of centrally located amorphous velop in relation to loose connective tissues and surmaterial. This material represents elastin as evidenced round vascular vessels and nerves, presumably funcby its TA-UA reactivity and removal with elastase di- tion to support these anatomical structures in a similar
gestion. The coexistence of the two types of small mi- capacity for functioning and nonfunctioning teeth.
The observation in the present study of HID-TCH-SP
crofibril bundles in the interstitial space may represent
consecutive stages of normal elastogenesis. The obser- staining in oxytalan- and elastic fiber-microfibrils folvation of a high ratio of the bundles with elastin t o lowing prior treatment of either monopersulphate or
those devoid of it may indicate relatively rapid elasto- cupric sulphite reagent but lack of the staining without
genesis in a given site. Thus the small microfibril bun- oxidation is consistent with previous studies demondles without elastin may correspond t o the earliest strating similar staining in monkey periodontal ligastages of elastogenesis rather than to a precursor form ments (Takagi et al., 1987) and mouse aortic tunica
in the development of large microfibril bundles or oxy- adventitia (Baba et al., 1988). This staining implies
talan fibers, which are relatively infrequent in the in- either the presence of disulphide linkages andtor sulterstitial space. This hypothesis is consistent with the phydryl groups or the absence of stainable sulphate in
previous observation that similar microfibril accumu- microfibrils as previously indicated (Takagi et al.,
lations occur prior to the onset of elastin deposition in 1987; Baba et al., 1988). The observation of the lack of
other elastic tissues of mammals (Karrer, 1958; Low, definitive HID-TCH-SP staining in the amorphous
elastin with and without oxidation is similar to that
1961; Ross, 1973).
The present and previous studies (Carmichael and observed in mouse aortic tunica adventitia (Baba et al.,
Fullmer, 1966; Fullmer et al., 1974; Cotta-Pereira et 1988). The typical HID-TCH-SP staining reaction of
al., 1976; Bock and Stockinger, 1984) have demon- oxytalan and elastic fiber microfibrils after oxidation is
strated ultrastructural and histochemical similarities extended into the lower vertebrates in the present
between oxytalan and elastic-associated microfibrils in study, thereby demonstrating a highly conserved stainthe Alligator and in mammals. This may relate to the ing pattern in the evolution of oxytalan and elastic
function of teeth and the stress of biting as Dreviouslv fibers in the Deriodontal ligament.
suggested (Fullmer, 1958; Fullmer et d.,
1674). Lig&
ACKNOWLEDGMENTS
microscopic studies (Soule, 1967) utilizing aldehyde
fuchsin with prior oxidation have demonstrated that
We wish to express our gratitude to Dr. Richard T.
oxytalan fibers in the Alligator periodontal ligament Parmley (University of Texas, Health Science Center,
are sparse in newly erupted teeth and are not seen San Antonio) for critical reading of the manuscript; Dr:
until the developing tooth is actively functioning. In Raymond F. Gasser (Louisiana State University) for
contrast, elastic fibers stained with orcein can be ob- providing fixatives; Ted Joanen, Ruth M. Elsey, and
served in the periodontal ligament of unerupted, non- the staff of the Rockfeller Wildlife Refuge, Grand Chefunctional teeth of the same animal (unpublished ob- nier, Louisiana, for their help; and Ms. Shoko Ogura
servations). Thus oxytalan fibers in collaboration with for her secretarial assistance.
Y
ALLZGATOIZ PERIODONTAL LIGAMENTS: ELASTIC ELEMENTS
LITERATURE CITED
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ultrastructure, fiber, elastica, periodontal, distributions, mississippiensis, differential, ligament, staining, alligator, oxytalan
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