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Electron microscopic studies on von Korff fibers in the human developing tooth.

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Electron Microscopic Studies on Von Korff Fibers in the
Human Developing Tooth
D. K. WHITTAKER AND D. ADAMS
Department of Oral Biology, Dental School, W e l s h National School of
Medicine, Heath, Cardiff, Wales
ABSTRACT
Deciduous tooth germs were removed from the anterior region
of human fetuses between 13-16 weeks of age and fixed in glutaraldehyde. Some
were post fixed in osmium tetroxide whilst others were block stained with silver
nitrate and prepared for electron microscopy. Thick Araldite embedded sections
were cut at 1 pm and stained in various ways for examination by light microscopy. Thin sections of bulk silver stained material were examined with the electron microscope before and after staining with heavy metals.
Fibers lying between the developing odontoblasts were demonstrable under the
light microscope and their appearance was consistent with the classical description of von KorfF fibers. With the electron microscope bundles of collagen fibers
up to 1 pm in diameter were present between newly differentiated odontoblasts.
Heavy metal staining of sections from bulk silver impregnated material revealed
that silver deposition was associated mainly with collagen fibrils. It is concluded
that in the crown of human developing teeth, bundles of collagen fibrils are
present between the newly differentiated odontoblasts and that these bundles
represent those usually described as von Korff fibers.
Similar results were found using material from developing molar teeth of mice.
Studies of mantle dentine of the developing tooth have included descriptions at the
light microscope level of prominent argyrophilic fibers coursing between the odontoblasts and terminating in the region of
the basement membrane separating these
cells from the internal enamel epithelium.
The earliest descriptions of these fibers by
Raschkow (1835) and Hansen (1899)
were amplified by von Korff ('05) whose
name has usually been applied to these
structures since that time. Although there
has been discussion regarding the presence
of von Korff fibers in later formed dentine
(Lester and Boyde, '68) their demonstration in forming mantle dentine using appropriate staining methods and light microscopy is in no doubt (Symons, '56).
With the electron microscope Masukawa
('59) illustrated a zone of "gnarled" collagen fibrils parallel to the odontoblastic
processes which might correspond to von
KorfF fibers. Scott and Nylen ('60) in a
description of the electron microscopic
features of dentinogenesis distinguished
between mantle dentine and circumpulpal
ANAT. REC., 174: 175-190.
dentine. They stated that in mantle dentine
there is a fan like arrangement of coarse
collagen fibrils which they designated as
the terminations of von Korffs fibers, while
in circumpulpal dentine the matrix was
composed of considerably finer fibrils arranged in the form of a network. Noble,
Carmicheal and Rankine ('62) demonstrated what they regarded as von Korf€
fibers in the area of the sheath of Hertwig
using human material while Johansen and
Parks ('62) and later Bevelander and
Nakahara ('66) described collagen bundles
up to 0.5 pm in thickness between newly
formed odontoblasts. Reith ('68) confumed the views of Scott and Nylen ('60)
and described the association of von Korff
fibers with odontoblasts that have just
begun to function in the developing molar
teeth of rats. He clearly showed bundles
of collagen fibers up to 1 pm in diameter
in the intercellular spaces of the odontoblasts and regarded them as representing
the classical fibers of von KorfF. Replica
technics and scanning electron microscopy
Received Nov. 3, '71. Accepted M a y 24, '72.
175
176
D. K. WHITTAKER AND D. ADAMS
led Lester and Boyde ('68) to confirm the
presence of collagen bundles in the mantle
dentine zone and these they regarded as
von Korff fibers.
On the other hand Lenz ('59) failed to
detect von Korff fibers as did Frank and
Nalbandian ('63). Takuma ('67) using
human material of 16-36 weeks intrauterine life was unable to distinguish fiber
bundles which he could regard as representing those described by von Korff. He
used no osmium fixation and only lead
hydroxide in the staining of his sections.
More recently Ten Cate, Melcher, Pudy and
Wagner ('70) reviewed many of these
earlier reports and stated that "no published electron micrographs of differentiating odontoblasts and early forming
dentine show bundles of collagen fibrils
sufficiently thick to be resolved as von
Korf€'s fibers in the light microscope." They
also presented evidence from their study of
mouse material to support their belief that
the classical appearance of these fibers in
the light microscope is due to silver impregnation of extracellular material and
not to deposition of silver on collagen
bundles.
Since these discrepancies may be due to
difficulties of nomenclature, to variations
between species or to differences in technic
or interpretation we have studied the differentiating odontoblasts of human tooth
germs using both light and electron microscopy with various staining technics and
sectioning procedures.
Some sections were stained with Toluidine
blue or Toluidine blue/malachite green,/
basic fuchsin (TMF) (Grimley, '64) whilst
others had the Araldite extracted and were
then stained with silver (Berkowitz,
Fiorello, Kruger and Maxwell, '68). Ultrathin sections were cut on the LKB ultratome. Those previously stained in osmiuni
tetroxide and embedded in Araldite were
stained on the grids with uranyl acetate
and lead citrate (Reynolds, '63) whilst
those impregnated with silver nitrate were
examined in the electron microscope and
photographed. They were then stained
with uranyl acetate and lead citrate and
the same or similar areas rephotographed
in the electron microscope.
For comparison we have also examined
the odontogenetic regions of molar teeth
from 1 day and 11 day old mice using
identical technics.
RESULTS
The area examined by the various technics is illustrated in figure 1. Predentine
can be distinguished and it was between
the underlying odontoblasts that collagen
fibers were sought. No structures distinguishable as fibers were seen in the Toluidine blue stained sections. However, in
those sections stained with TMF, magenta
stained fiber-like structures of 1 pm diameter were seen in the extracellular spaces
of the odontoblasts (fig. 2). These were
at the same site as fiber-like structures in
silver impregnated blocks cut at 2 /.mi
(fig. 3). Magenta stained fibers were also
MATERIALS A N D METHODS
seen randomly arranged in the zone beAnterior tooth germs were excised from tween the odontoblasts and the basement
human fetal material of 13-16 weeks ob- membrane in TMF stained sections and
tained at abdominal hysterotomy and fixed these corresponded to the dense silver
in 3% glutaraldehyde (Karlsson and stained area at the same site in silver
Schultz, '65) at 4°C for one to two hours. impregnated blocks.
The tooth germs were either post-fixed
Sections of oral mucosa overlying the
in osmium tetroxide or impregnated with developing tooth germs stained in the
silver nitrate according to the technic em- block by silver impregnation also showed
ployed by Ten Cate et al. ('70). Oral structures of similar dimensions near the
mucosa overlying the tooth germs was basement membrane and lying between
similarly prepared and all tissues were fibroblasts.
embedded in Araldite. Thick sections (0.5
Electron microscopical examination of
,m-l /.m) were cut on the LKB pyrami- sections adjacent to those examined in the
tome and orientated in such a way that the light microscope confirmed in all cases
inner enamel epithelium and early odonto- that the fiber like structures present beblasts were sectioned at right angles to tween differentiating odontoblasts and
the basement membrane between them. near the basement membrane of oral
COLLAGENOUS NATURE OF VON KORFF FIBERS
mucosa were banded collagen fibrils
arranged in bundles between 0.5 ,m and
1 p m in diameter. These bundles were
composed of fibrils showing the regular
640 A banding of collagen arranged in
groups of between 8 and 14 single fibrils.
Some of the bundles so formed were sectioned longitudinally and could be followed over a length of up to 5 pm (fig. 4).
Other bundles of similar diameters were
cut transversely. Although the fibers were
lying freely in the spaces between the
odontoblasts they were in some situations
very closely associated with the plasma
membrane of the odontoblasts. In the
space between the basement membrane
and the odontoblasts randomly arranged
collagen fibrils were also visible.
The sections from tissues impregnated
in the block with silver showed a deposition of silver granules up to 500 A diameter which were located in the intercellular spaces. This was so both in the case
of odontoblasts (fig. 5a) and in the connective tissue below oral epithelium (fig.
6a). Silver granules were deposited in the
area between the lamina densa and the
differentiating odontoblasts (fig. 7) and
this deposition was related to the randomly arranged collagenous fibers seen in
figure 4.
The same sections when overstained
with heavy nietals clearly showed that the
silver grains were mainly deposited where
collagen bundles were present (figs. 5b,
6b). The relationship of silver grains to
collagen fibrils and their scarcity where
no collagen was seen can be clearly noted.
Similar results were found using material
from 1 day and 11 day old mouse molar
teeth.
DISCUSSION
In this discussion a collection of individual elements of collagen fibrils will be
referred to as a collagen bundle in accordance with the terminology of Melcher and
Eastoe ('69). The results of our light microscopical study on thick sections stained
with both silver and TMF have shown
that fibers can be easily resolved in the
spaces between odontoblasts associated
with early dentinogenesis.
Our electron microscopical findings confirm the results of Reith ('68) and other
177
authors that collagen bundles are present
in the site at which light microscopists
have described the von Korff fibers. The
argument remaining is whether these are
resolvable in the light microscope and of
the same order of size as classically described von Kofl fibers and secondly
whether silver impregnation in the block
does in fact stain these structures or the
ground substance surrounding them.
Many of the bundles visible on our electron micrographs are fan shaped and 0.8
p m at the narrow end, widening to 1.0
at the widest end. Structures of this diameter should be easily resolvable in the light
microscope and approach in diameter the
silver impregnated areas usually described
as von Korff fibers in the light microscope
(Tomes, 1849, Symons, '56; Ten Cate
et al., '70). Moreover deposition of silver
on such structures would tend to facilitate
their resolution in the light microscope.
Ultra-thin sections for the electron microscope only rarely will pass through the
maximum diameter of such bundles and
this may explain the somewhat narrower
bundles described by some workers (Nylen
and Scott, '60). For similar reasons the
length of up to 5 pm is compatible with
somewhat longer structures pursuing a
less than straight course and sectioned
only at intervals along their length. The
fact that some workers (Takuma, '67; Ten
Cate et al., ' 70), have been unable to demonstrate these fibers clearly may be due
to staining technics or to unfavourable
section plane.
Heavy metal staining of areas that have
been impregnated with silver clearly suggest that the silver is in fact deposited on
or immediately around collagen fibrils.
This is so not only in association with the
von Korff fibers but also in other areas
where collagen is present such as the connective tissue underlying developing oral
mucosa. Photographs of the intercellular
areas of the odontoblasts by Ten Cate et al.
('70) do show some slight evidence of collagen fibrils in these sites but since no micrograph following heavy metal staining is
presented for comparison it seems that the
possibility of collagen bundles in these
situations still exists in the developing
mouse tooth germ. In fact, we have found
no differences in fiber deposition in this
178
D. K. WHITTAKER AND D. ADAMS
site when mouse and human material Nagai ('71) their material was from the
lingual side of the rat incisor and therefore
were compared.
Silver staining is unpredictable and not underlying enamel. The fibers which
probably depends on fixation technics, Noble et al. ('62) described and referred
buffer systems used and the exact method to as von Korff fibers are smaller and
of application of the staining reaction. It is sparser than those described here and it
difficult for these reasons to compare is noteworthy that their material was from
results of different workers but the same the root end of a developing tooth.
The third possibility that the fibers are
technics of fixation, washing, embedding
and staining have been used in the present associated with the orientation of the prework for both light and electron micros- odontoblasts in a direction at right angles
copy. We therefore feel justified in stating to the melo-dentinal junction also has to
that the silver stain visible between differ- be considered. They may give some degree
entiating odontoblasts in our light micro- of support to the rather scattered cells
scope studies is in fact related to collagen prior to the formation of the regular layer
or precollagenous reticulin at this site and of odontoblasts with desmosomal junctions
is in the position believed to be occupied between neighbouring cells. Once the iniby von Korff fibers. In the present context tial layer of dentine and enamel have been
the argument as to whether the fibers are elaborated there is less chance of the discollagenous or precollagenous reticulin is ruption of the odontoblast layer and so
not really relevant since our main aim is von Korff fibers no longer are required.
a correlation of silver staining material in They are then incorporated in the dentine
giving it sufficient distingushing features
light and electron microscopy.
The function of the von KO& fibers is to enable it to be called mantle dentine.
still in doubt. Most writers who described It would seem to be of value to study the
them are dealing with the earliest formed initial production of dentine in animals
dentine, i.e., that dentine which lies under made lathrytic where collagen would be
the enamel. If these fibers do have a func- deficient but more work is required to detion, therefore, it seems reasonable to as- termine the significance of this peripheral
sociate them with the amelo-dentinal junc- layer of dentine.
In relation to early dentine formation it
tion, with the physical properties of the
mantle dentine, or with the odontoblasts is of interest to note that the 5 pm wide
zone of randomly arranged collagenous fithemselves.
The f i s t possibility seems unlikely since bers lying between the lamina densa and
the spacing between von Korff bundles is the differentiating odontoblasts was also
much less than the periodicity of scallop- silver positive and was stained magenta by
ing of the amelo-dentinal junction and TMF. A comparison of this zone in figures
moreover no evidence has been produced 2, 3, 4 and 7 clearly indicates the site of
in this or previous work, that they are silver stained material and the relationships of this silver to the collagenous fibers.
inserted into the lamina densa.
The second possibility that they con- This staining reaction in the early dentinal
tribute to the physical properties vf the matrix is in accord with that seen in the
mantle dentine seems more likely. Lester case of the von Korff fibers and the connecand Boyde ('68) remark on the expected tive tissue fibers of the oral epithelium and
rigidity and mechanical stability of a von supports our view of the fibrous nature of
Korff bundle and relate this to a pressure von KO& fibers.
build up during proliferation of the dental
The conclusions that we draw from this
pulp. The contraction and distortion of work suggest that the classically held view
enamel during maturation (Starkey, '71 ) of the collagenous nature of von Korff
may also require a rigidity of the mantle fibers is correct. Collagen bundles of up
dentine area which could be provided by to 1.0 pm in diameter and 5.0 pm in length
these fibers. It is interesting in this con- have been demonstrated at the electron
nection to note that although von Korff microscope level at the site usually befibers appeared to be absent in a recent lieved to be occupied by von Korff fiber
study of rat odontogenesis by Takuma and bundles. These bundles were stained with
COLLAGENOUS NATURE OF VON KORFF FIBERS
silver both by impregnation and by staining of Araldite sections and were positive
with another stain for collagen. The silver
deposited extracellularly in block stained
material was associated with collagen
bundles in om material.
ACKNOWLEDGMENTS
Our thanks are due to Professor B. E. D.
Cooke for his helpful criticism and advice
on this paper. We are also grateful to Professor A. Turnbull and his staff for the
material used in this study. The technical
assistance of Mrs. C . Winters and
Mr. R. Watkins is gratefully acknowledged.
LITERATURE CITED
Berkowitz, L. R., 0. Fiorello, L. Kruger and D. S.
Maxwell 1968 Selective staining of nervous
tissue for light microscopy following preparation for electron microscopy. J. Histochem.
Cytochem., 16: 808-814.
Bevelander, G., and H. Nakahara 1966 The
formation and mineralisation of dentin. Anat.
Rec., 156: 303-323.
Frank, R. M., and J. Nalbandian 1963 Comparative aspects of development of dental hard
structures. J. Dent. Res., 42: 422437.
Grimley, P. M. 1964 A tribasic stain for thin
sections of plasticembedded Os04-fixed tissues.
Stain Tech., 39: 229-233.
Hansen, F.C. C. 1899 Uber die Genese einiger
Bindegewebsgrundsubstanzen Anat. Anz., 16:
4 17-438.
Johansen, E., and H. F. Parks 1962 Electron
microscopic observations on sound human
dentine. Arch. oral Biol., 7: 185-193.
Karlsson, U., and R. Schultz 1965 Fixation of
the central nervous system for electron microscopy by aldehyde perfusion. J. Ultrastruct. Res.,
12: 160-186.
KO&, K. Von 1905 Die Entwicklung der Zahnbeingrund-substanz der Saiigetiere. Arch. mikr.
Anat., 67: 1-17.
I,enz, H. 1959 Electronenmikroskopische Untersuchungen der Dentinentwicklung. Dtsch. Zahn
Mund-u. Kieferheilk, 30: 367-381.
179
Lester, K. S., and A. Boyde 1968 The question
of von KorE fibres in mammalian dentine. Calc.
Tiss. Res., I: 273-287.
Masukawa, K. 1959 Electron microscopic study
o n the ultrastructure of predentine. Bull. Dept.
Anat. Oska dent. Coll. No. 20. pp. 1-7.
Melcher, A. H., and J. E. Eastoe 1969 T h e connective tissues of the periodontium. In: The
Biology of the Periodontium. A. H. Melcher and
W. H. Bowen, eds. Academic Press, London,
New York, pp. 17G343.
Noble, H. W., A. F. Carmicheal and D. M.
Rankine 1962 Electron microscopy of human
developing dentine. Arch. oral Biol., 7: 395-399.
Nylen, M. U., and D. B. Scott 1960 Electron
microscopic studies of odontogenesis. J. Indiana
dent. Assoc., 39: 406-421.
Raschkow, I. 1835 Meletemata circa mammalium dentium evolutionem. Thesis: M. Friedlaender , Vr atislaviae.
Reith, E. J. 1968 Ultrastructural aspects of
dentinogenesis. In: Dentine and Pulp. N. B. B.
Symons, ed. E. S. Livingstone Ltd., Edinburgh
and London, pp. 19-57.
Reynolds, E. S. 1963 The use of lead citrate
at high pH as a n electron opaque stain i n electron microscopy. J. cell Biol., 17: 208-212.
Scott, D. B., and M. U. Nylen 1960 Changing
concepts in dental histology. Ann. N. Y. Acad.
Sci., 85: 133-144.
Starkey, W. E. 1971 Dimensional changes associated with enamel maturation in rabbits.
Arch. oral Biol., 16: 479-493.
Symons, N. B. B. 1956 The development of
the fibres of the dentine matrix. Brit. Dent. J.,
101: 252-262.
Takuma, S. 1967 Ultrastructure of dentinogenesis. In: Structural and chemical organisation of teeth. A. E. W. Miles, ed. Academic
Press, London and New York, pp. 325-370.
Takuma, S., and N. Nagai 1971 Ultrastructure
of rat odontoblasts in various stages of their
develoument and maturation. Arch. oral Biol..
16: 993-1011.
Ten Cate, A. R., A. H. Melcher, G. Pudy and
D. Wagner 1970 The non-fibrous nature of
the von K o f l fibers in developing dentine. A
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Abbreviations
LD, Lamina densa
DO, Differentiating
odontoblasts
IEE, Internal enamel
epithelium
PD, Predentine
VK, Von Korff fibers
PLATE 1
EXPLANATION OF FIGURES
1 Light micrograph of 1 pn section of human anterior tooth germ.
Electron micrographs were prepared from the area where differentiating odontoblasts are present. Araldite embedded and stained with
toluidine blue. x 500.
180
2
Region of differentiating odontoblasts photographed under oil immersion. Fibrous structures (arrowed) are visible between the odontoblasts
and r u n perpendicular to the basement membrane. Araldite embedded,
and stained with toluidine blue/malacite green/basic fuchsin. Facing
black arrows o n this and subsequent figures indicate zone of randomly arranged collagen fibrils between lamina densa and differentiating odontoblasts. x 1200.
3
Comparable area to that shown in figure 2. Fiber-like structures can
be seen in the intercellular area of the odontoblasts. Bulk silver
impregnated and Araldite embedded. x 1000.
COLLAGENOUS NATURE OF VON KORFF FIBERS
D. K. Whittaker and D. Adams
PLATE 1
181
PLATE 2
EXPLANATION O F F I GU R E
4
182
Electron micrograph of newly differentiated odontoblasts in the
human tooth germ. Collagen fibrils are collected into bundles u p to
1 pm in diameter lying in the intercellular spaces and orientated at
right angles to the basement membrane. Stained with osmium
tetroxide, Araldite embedded and post-stained with uranyl acetate
and lead citrate. x 10,000. Insert shows silver impregnated section,
under light microscopy, of similar region.
COLLAGENOUS NATURE OF VON KORFF FIBERS
PLATE 2
D. K. Whittaker and D. Adams
183
PLATE 3
EXPLANATION O F FIGURES
184
5a
Early dentinogenesis in a human tooth germ impregnated with silver.
Granules of silver c a n be seen i n the intercellular space of the
odontoblasts. These are associated with the vague outlines of fibrous
material. Bulk silver impregnation. Osmium tetroxide staining and
Araldite embedded. x 33,000.
5b
Adjacent section to figure 6a showing the same area following heavy
metal staining. Collagen fibrils arranged in bundles can be seen
associated with the silver granules. Bulk silver impregnation followed
by uranyl acetate and lead citrate staining. Araldite embedded
x 33,000.
COLLAGENOUS NATURE OF VON KORFF FIBERS
D. K. Whittaker and D. Adams
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
186
6a
Fibroblasts deep to oral epithelium overlying a tooth germ. Silver
granules are present between the adjacent cells. Bulk silver impregnation and osmium tetroxide staining. Araldite embedded. x 15,000.
6b
Adjacent section to figure 6a showing the same area following heavy
metal staining. The relationships of silver granules to collagen fibrils
is now revealed. Bulk silver impregnation and osmium tetroxide staining followed by uranyl acetate and lead citrate and Araldite
embedded. X 15,000.
COLLAGENOUS NATURE OF VON KORFF FIBERS
PLATE 4
D. K. Whittaker and D. Adams
187
PLATE 5
EXPLANATION OF FIGURE
7 Human tooth germ impregnated with silver. A zone of silver granules
approximately 5 Fm in width (indicated by black arrows) is clearly
visible between lamina densa of differentiating odontoblasts. This
zone and the silver granules on von Korff fibers should be compared
with the similar zone in figures 2, 3, and 4.
188
COLLAGENOUS NATURE OF VON KORFF FIBERS
D. K. Whittaker and D. Adams
PLATE 5
189
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