Evidence for a Junctional Epithelial Attachment to

Ceramic Dental Implants*

A Transmission Electron
Ralph

V.

McKinney, Jr.,f

David E.

Microscopic Study
Steflikf and David

Accepted for publication 18

L.

Kothij:

March 1985

with the surface of a dental implant is a critical

which protects the underlying bone and soft
the
seal
representing
potential biological
tissue-supporting mechanisms from destructive extraneous substances. Ultrastructural examination of regenerated junctional epithelial cells interfacing surgically placed endosteal
dental implants, comprised of alpha-alumina oxide ceramic in single crystalline form,
exhibited an external basal lamina and linear body located between the external surface
epithelial cell and the implant. In addition, hemidesmosomes were located at intervals along
the outer junctional epithelial plasma membrane. The component substructures of the basal
lamina and the hemidesmosomes were similar to those seen interfacing natural teeth. The
linear body was an electron-dense structure between the lamina densa and the inert
biomaterial. This study provides ultrastructural evidence for the presence of an attachment
complex between gingiva and aluminum oxide implants which is analagous to that seen
around natural teeth. These data support the concept that a viable biological seal can develop
around endosteal dental implants and provide support for satisfactory clinical service.
The

interface of the crevicular gingiva

zone

Modern endosteal dental implants have been used

for approximately 45 years as a treatment for the replacement of missing teeth in the partially or completely edentulous patient. A critical feature that is
common to all dental implant designs and materials is
the status of the soft tissue-dental implant interface.
This transmucosal passage of the implant from the
internal aspects of the jaw bone and soft tissue into the
external aspects of the oral cavity is a situation unique
to dental implantology and makes the biological criteria
for dental implants different from implants used in
orthopedic surgery. For the dental implant to survive
in this dual environment, a viable biological seal that
prevents the ingress ofbacteria and other inflammationproducing agents must exist at the soft tissue-implant

Methodology has improved considerably over relight microscopic examination of the

gingival-biomaterial interface has become practical.3"6
There are currently a number of reports detailing the
histological appearance of the gingival adaptation to a
protruding implant coronal post.7"11
A few workers have been able to prepare peri-implant-tissue specimens for examination by scanning and
transmission electron microscopy. McKinney and coworkers1213 have shown with scanning electron microscopy that the regenerated gingiva forms a viable cuff
around the coronal implant post and extends epithelial
pseudopodia from the junctional epithelium, similar to
that seen interfacing natural teeth, that contact the
biomaterial face. However, the scanning electron microscope resolution prevents determining the exact nature of the cell organdes that are involved in the
interface biology between epithelial cells and the implant material.12
als.

cent years, and

interface.12

Examination of this biological seal is a difficult technical problem because it requires manipulating soft and
hard tissues simultaneously with implanted biomateri-

The first in vivo ultrastructural demonstration of

hemidesmosomes and a basal lamina between regenerated junctional epithelia and a Vitallium dental im-

supported in part by Grant No. 7834-0001 from

Kyocera, International, Inc. and Grant No. 1538-00-04 from Johnson
*

This

study

was

and Johnson, Inc.

t Department of Oral Pathology, Medical College of Georgia,
Augusta, GA.
Department of Fixed Prosthodontics, University of North Carolina at Chapel Hill, Chapel Hill, NC.

plant was presented by James and Schultz14 using transmission electron microscopy. This was followed by the
demonstration of Listgarten and Lai of hemidesmosomes and a basal lamina against epoxy resin dental

579

580

S. Periodontol.
October, 1985

McKinney, Stefiik, Koth

endosseous implants in vivo.15 Most recently, others

have shown the presence of epithelial cell adhesion
organdes against titanium alloy (T-6A1-4V) and pure
titanium in vitro16 and in v/vo.1718 Swope and James19
found that these attachment organdies form as early as
24 hours after Vitallium implant insertion.19
The purpose of this investigation was to examine,
using transmission electron microscopy, the cells that
interface with an endosseous dental implant comprised
of -alumina oxide ceramic material. The evidence of
cellular activity and organdies similar to that seen in
the normal tooth-soft tissue interface would be strong
evidence to support the suggestion that a viable biological seal exists around implanted ceramic materials and
would further denote the potential for long-term serviceability of these dental implants.
MATERIALS AND METHODS

The

for periods up to 24 months. The dogs were clinically

evaluated every 3 months using a standardized protocol
which allowed statistical interpretation of the data.
Results of these clinical trials and their statistical interpretation have been recently reported.24'25
At the beginning of the experiment, randomly selected animals were scheduled for sacrifice and microscopic examination. Some of the recovered specimens
were used for light microscopy, some for scanning
electron microscopy, and some for transmission electron microscopy. Five animal specimens from 9, 12, 18
and 24 months were selected for high resolution transmission electron microscopy (TEM).
Prior to TEM specimen preparation, the animal
heads were fixed by vascular perfusion employing either
3% phosphate buffered glutaraldehyde or 10% neutral
buffered formalin following a carotid artery cut-down
procedure. For perfusion, the fixative was administered

by a peristolic pump at 175 cc/minute at approximately

80 mm of Hg for a period of 45 minutes. The external

single-crystal sapphire dental implant is a relatively new implant material introduced into the United
States after initial investigative studies in Japan.2021
The material consists of crystalline, -alumina oxide
prepared in the configuration of a cylindrical screw
(Fig. 1). The physical characteristics of the implant
material and its various designs have been reviewed.22
For this study, eighteen 20- to 25-kg mongrel dogs
had single-crystal sapphire dental implants placed in an

jugular veins were severed to allow escape of the blood

and perfusate during the procedure.
Following this initial fixation, the mandibles containing the implants fixed by 10% neutral buffered formalin
were block-resected, and the portion containing the

by the extraction of the third and fourth premolars 8

weeks previously. The methods of animal preparation,
implant surgical insertion and follow-up clinical evaluation data have been covered in previous reports.1 K23'24
Thirty-six single-crystal sapphire implants were placed
bilaterally in the mandibles of these dogs.
The implants were left in place in the animal jaws

methyl methacrylate (PMMA) according

edentulous zone of the mandible that had been created

Figure 1. A photograph of the single crystal sapphire endosteal dental

implant composed ofa-alumina oxide ceramic material. The implants
are available in several lengths and widths which allow for selection
of an appropriate size for the recipient host. The implant is extremely
smooth on its outer surface.

in situ was re-immersed in 10% formalin for

48 hours. Upon completion of fixation, these block
samples were washed in 0.1 m phosphate buffer (pH
7.4) and dehydrated through a series of graded ethanols
of 50%, 70%, 95%, 95%, 100%, 100% for 12 hours at
each stage. The samples were then embedded in poly-

implant

to our

pub-

lished procedure6 and were sectioned buccal-lingually

with a Buehler Isomet saw and diamond wafering blade
at either 120 or 2 to 5 mm.
The heads that were perfusion-fixed with 3% phosphate buffered glutaraldehyde were processed differently; following the initial 45-minute perfusion schedule, small 3x4 mm blocks of the free gingiva interfacing the implant were carefully microdissected away
from the implant and reimmersed in 3% glutaraldehyde
for a total fixation time of 4 hours. The tissue dissection
was accomplished by fine surgical instruments, magnifying loops and careful attention to tissue orientation.
Following the 4-hour fixation time, the gingival specimens were cubed to 1 mm3 for further TEM processing.
Again, critical attention to tissue orientation was observed. The samples were washed three times (20 minutes each wash) in 0.1 m phosphate buffer and postfixed for 2 hours in 1 % osmium tetroxide buffered to
pH 7.4 with 0.1 m phosphate buffer. After post-fixation,
the samples were again washed, then passed through a
graded series of ethanols of 50%, 70%, 95%, 95%,
100%, 100%, 100% for 10, 15, 20, 20, 30, 30, 30
minutes, respectively. These samples were transferred
through propylene oxide and embedded in Epon 812
according to Luft's method.26

Volume 56
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Junctional Epithelial Attachment to Ceramic Implants 581

The microdissected, Epon-embedded specimens were

thin-sectioned using an LKB Ultratome III and glass
knives. Orientation sections were cut at 1.0 , and
thin sections were cut at approximately 75 nm.
Because of the concern that the microdissection procedure may have disrupted structures external to the
epithelial cell unit membrane, a cryofracture technique
was developed using our PMMA-embedded specimens.
For the cryofracture procedure, the 2 to 5 mm PMMA
sections were frozen in liquid nitrogen for 30 seconds
followed immediately by immersion in boiling distilled
water. This procedure resulted in the soft tissue fracturing away from the implant. The fracture plane occurred
along the implant-organic tissue interface because of
structural differentials. The recovered cryofractured
gingival specimens were then reembedded in PMMA
and polymerized. Orientation was simplified by the fact
that the sections were previously histologically stained
thus providing anatomical markers.27
Thin sectioning was again accomplished with an LKB
Ultratome III with orientation sections stained in 1%
toluidine blue and thin sections stained with uranyl
acetate and lead citrate.
All TEM sections were viewed with either an RCA
EMU4C or a Philips 200 transmission electron microscope.

RESULTS

Our transmission electron microscopic studies used

methodologies to obtain high resolution information of the critical soft tissue-implant interface, a microdissection technique and a cryofracture technique.
To organize the results in a logical manner, we present
first the data obtained from microdissection, followed
by the cryofracture technique.
Microdissected TEM Specimens. Low power electron
microscopic views of the overall crevicular cell layer
provided orientation to the nature of the junctional
epithelium which was found to be 3 to 7 cells thick.
The internal (or basal) layer of junctional epithelial cells
revealed normal intercellular junctions, a prominent
basal lamina exhibiting both lamina lucida and lamina
densa substructure, and anchoring fibrils which were
associated with the underlying collagen fibers (Fig. 2).
The external surface layer of junctional epithelial cells
demonstrated normal cytoplasmic organelles: nucleus,
tonofilaments, Golgi, ribosomes and endoplasmic reticulum (Figs. 2 and 3). The outermost plasma membrane
that interfaced with the implant was primarily flat, but
did contain at various intervals cytoplasmic pseudopodia (Fig. 2a). The outer epithelial cells also showed the
presence of various flocculent densities in association
with the membrane (Fig. 3). At various intervals along
the outer cell surface, dense laminated organelles were
associated with the plasma membrane (Figs. 3 and 4).
These laminated organelles, or hemidesmosomes, were
two

present on flat portions of the cell membrane as well as

on the membrane of pseudopodial projections emanating from the cell (Fig. 5). Desmosomes were prominent
between contacting intercellular bridges of the junctional epithelial cells (Fig. 2) and were similar in density
and structure to the hemidesmosomes noted on the
outer plasma membranes.
Fine filaments extending from the body of the hemidesmosome structure into the cytoplasm of the cell
observed (Fig. 4). At very high resolution, the
laminated substructure of the hemidesmosome was
clearly revealed (Fig. 6).
The outermost cells of junctional epithelium contained a rich population of membrane-bound secretory
vesicles (Figs. 7 and 8). These vesicles were prominent
throughout the cell cytoplasm including association
with Golgi (Fig. 9), but appeared more densely aggregated at or near the implant front unit membrane (Figs.
7 and 8). Some of the vesicles gave the appearance of
coalesced aggregates at the implant front (Fig. 7), and
vesicles often were aligned with the outer plasma membrane (Fig. 8). Occasionally, fusion of the vesicular
membrane with the outer plasma membrane was observed suggesting an opening for the discharge of the
vesicle contents externally (Fig. 10).
A varied population of vesicles was observed in the
junctional epithelium at high resolution: single unit,
membrane-bound vesicles and double unit, membranebound vesicles (Fig. 11 ). Both of these type of vesicles
were found intimately related with endoplasmic reticulum and Golgi (Figs. 9 and 11 ). Very high magnification of other vesicles demonstrated a central dense
arrangement of contents surrounded by an outer lucent
zone containing dense strands of material (Fig. 12).
Some sections revealed secretory vesicles that were
irregular in shape and contained electron dense flocculent material of varying density that was similar in
morphology to material found on the outer unit membrane (Fig. 13). Tonofilaments were present in varying
numbers in the junctional epithelium (Figs. 3, 7, 8 and
were

13).
Cryofractured TEM Specimens. The cryofractured
junctional epithelium revealed adequate morphological
retention of cellular architecture with clearly distinguishable nuclei, nuclear envelopes, nucleoli, intercellular bridges, desmosomes, ribosomes, endoplasmic reticulum and mitochondria in spite of the formalin
fixation and double PMMA embedding procedures
(Fig. 14). Some artifactual disruption of the cell sap was
evident at high magnification, but cell membranes and
their attendant structures remained intact (Fig. 16).
Ultrastructural examination of the most superficial
junctional epithelial cells interfacing the implant revealed a clearly distinguishable unit membrane, an
external basal lamina with lamina lucida and lamina
densa components, and clearly definable hemidesmosomes (Figs. 15, 16 and 17). The lamina densa and

582

McKinney, Stefiik, Koth

J. Periodontol.

October, 1985

Figure 2. Low power Iransmission electron micrographs of the regenerated junctional epithelium that was microdissectedfrom around a sapphire
implant, a. The external surface junctional epithelial cells interfacing the implant (space at top left contained implant) demonstrate intercellular
bridges with desmosomes (d), intercellular spaces (*), nucleus (N), surface pseudopodial projections (arrowheads) and two polymorphonuclear
leukocytes (P) which are frequently seen in junctional epilhelia. Present throughout the two cells are electron dense secretory vesicles (V), that are
aggregated in greater numbers at the external unit membrane of the epithelial cell interfacing the implant (magnification x 8000). b. Basal cells
of the junctional epithelium exhibit intercellular bridges (arrowheads), desmosomes (d), intercellular spaces (*), nucleus (N) and the lamina lucida
(11) and lamina densa (Id) components of the basal lamina. Anchoring fine filaments (ff) extend from the basal lamina into the supporting
connective tissue around collagen fibers (c). Hemidesmosomes (h) appear as dense plaques on the cell membrane interfacing the basal lamina
(magnification x 18,000).


Figure 3. transmission electron micrograph of an interfacing junclional epithelial cell in a microdissected gingival specimen. The implant occupied the space at the top left. Organelles are prominent
throughout the cell and include the nucleus (N), rough endoplasmic
reliculum (er), free ribosomes and numerous tonoftlaments (tf). Not
present in this particular cell are the numerous secretory vesicles seen
in Figure 2a. The external plasma membrane exhibits a dense plaque
structure suggestive of a hemidesmosome (h) and scattered flocculent
deposits (arrowheads) associated with the membrane (magnification
X 10,400).

Figure

5. Three

prominent hemidesmosomes (arrows) present

on a

pseudopodial projection extending from an interfacing junctional ep-

ithelial cell. External basal lamina structure is not visible in this

surgically dissected specimen (magnification x 38,750).

WBSKmM

Figure 4. A hemidesmosome (h), located on the external unit memof an interfacing epithelial cell exhibits a fine filamentous
process emanating from the hemidesmosome into the cell cytoplasm
(arrowheads). Note the variable density of the vesicle contents (V) in
this microdissected gingival specimen. The cell cytoplasm and nucleus
(N) are slightly out offocus since fine focusing was concentrated on
brane

Figure 6. High resolution transmission electron micrograph revealing

the subunit structures of a hemidesmosome on the interfacing unit
membrane of a microdissected specimen. The implant occupied the
space at the figure top. Visible is the unit membrane (urn), peripheral
bar or density (pd), pyramidalparticles (below dots) on unit membrane
and finefilaments (arrowheads) extendingfrom the hemidesmosome.
An external basal lamina is not demonstrated (magnification x
177,500).
the hemidesmosome, filamentous process and the vesicle. The implant
was located at the top of the picture (magnification x 23,400).

584

McKinney, Stefiik, Koth

J. Periodontol.
October, 1985

Figure 7. Transmission electron micrograph of microdissected junctional epithelial specimen removed from around the implant. Numerous
secretory vesicles (V) are aggregated at the implant front near the outer unit membrane (lop of micrograph) in the first tier ofepithelial cells (1).
Fewer vesicles occur in the second tier (2) of epithelial cells. Desmosomes are present between epithelial projections (arrowheads) (magnification
x

18,900).

Figure 8. Transmission electron micrograph of microdissected junctional epithelial specimen removed from around the implant. Secretory vesicles
(V) containing dark material are aligned along the implant front of an interfacing junctional epithelial cell. Two vesicles appear to have coalesced
with the unit membrane (arrows) (magnification x 25,200).

Volume 56
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Junctional Epithelial Attachment to Ceramic Implants 585

Figure 9. Transmission electron micrograph of microdissected functional epithelial specimen removedfrom around the implant. Secretory
vesicles of varying density, and rihosomes surround a Golgi (G) in a
junctional epithelial cell (magnification x 25,200).

12. Transmission electron micrograph of microdissected junctional epithelial specimen removed from around the implant. Two
epithelial vesicles which exhibit dense center zones surrounded by a
distinctive outer rim, all contained in a single unit membrane (mag-

Figure

nification x 92,300).

revealing an internal complex organization (Figs.

18). At times the outer limit of the lamina densa
flocculent
and slightly indistinct in its outline (Figs.
was
Close
examination of the lamina densa
and
17
18).
the
presence of two distinct electron-dense
suggested
and 18). At very high resolution, a
17
layers (Figs.
ture

17 and

electron-lucent band was observed between the

lamina densa and the outermost electron-dense layer
narrow

(Fig. 18).
High magnification electron microscopy also allowed
Figure 10. Transmission electron micrograph of microdissected junctional epithelial specimen removed from around the implant. High
resolution micrograph of a membrane-hound secretory vesicle fused
with the epithelial plasma membrane at the implant front and revealing

an

opening

to

the external environment. Vesicular content disby the presence of similar dense material within

resolution of the substructure of the hemidesmosomes

to include the peripheral densities, pyramidal particles
and fine filaments associated with the unit membrane
and basal lamina (Figs. 6 and 15-18).
DISCUSSION

Figure 11. Transmission electron micrograph of microdissected junctional epithelial specimen removed from around the implant. High
resolution electron micrograph showing a double unit membrane
vesicle (double arrow) and a single membrane-bound vesicle (single
arrow) with contents of variable density, near a portion of Golgi (G)
(magnification x 46,000).

Dental implants have increased in sophistication

since their modest modern beginnings in the early
1940s. The literature contains numerous articles concerning implant longevity, clinical serviceability, function and even rudimentary statistical analyses of human
cases.28'29 However, very little attention has been accorded the biological interface between hard and soft
tissues and the implanted material. The fact that implants do function for extended periods of time and
provide adequate and satisfactory service to many patients raises the question as to what causes or creates
an acceptable environment in the human oral cavity.
This investigation conclusively demonstrates that regenerated crevicular gingiva around ceramic dental implants develops junctional epithelial organelle and cellular architecture that is similar to the morphological
structures that interface the natural tooth.30"36 The presence of these structures adjacent to the implant face
provides biological evidence for successful long-term
clinical service of the ceramic endosseous dental im-

lamina lucida varied in width along the implant front

with the most variance being shown by the lamina
densa (Figs. 15-18). The lamina densa varied from an
even width, electron-dense structure to a varied struc-

plants.
Secretory Vesicles. Our initial experimentation with
the microdissected specimens showed the presence of
junctional epithelial cells and their organdes as described by Schroeder.32 We thus expected to find in the

charge

is indicated

the vesicle and extra cellularly (arrow)

(magnification x 44,800).

586

McKinney, Stefiik, Roth

J. Periodontol.

October, 1985

A junctionai epithelial cell dissectedfrom the implant (space, upper left) containing two vesicles (V) of varying shape and morphological
The density of the flocculent material along the outer unit membrane (arrows) is similar to that observed in the vesicles, although no
organized basal lamina is visible along this microdissected specimen cell surface. Tonofilamenls are prominent in the cytoplasm (tf) (magnification

Figure 13.
contents.
x

82,000).

Figure 14. The basal cell layer of junctionai epithelium from a formalin-fixed, PMMA-embedded specimen that was reprocessed for the
cryofracture technique (see text). Note the adequate retention of overall cell morphology incuding nucleus (N), nucleolus (Nu), ribosomes (r), rough
endoplasmic reticulum (er), intercellular bridges (arrows) and intact desmosomes (d) (magnification x 42,500).

Volume 56
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Junctional Epithelial Attachment to Ceramic Implants 587

Figure 15. High resolution transmission electron micrograph of the outer junctional epithelial cell unit membrane. The unit membrane interfacing
the implant demonstrates periodic hemidesmosomes (h) composed of peripheral densities and pyramidal particles (pd) with fine filaments (if).
The basal lamina substructure of lamina lucida and lamina densa is very distinct, although the lamina densa varies in width (between arrowheads)
as well as outer surface morphology. This specimen was formalin-fixed, PMMA-embedded then processed for the cryofracture technique and
reembedded in PMMA. The implant occupied the top space in this figure (magnification x 89,700).

Figure 16. This section of an epithelial cell membrane reveals a lamina lucida (11) wider than the lamina densa (Id). Hemidesmosomes (h) on the
unit membrane demonstrate distinct peripheral densities with pyramidal particles (below dots). Fine filaments emanate from the density into the
lamina lucida and cell cytoplasm. The nature of the surface dense particle is unknown. Processing artifact most likely is present at the asterisks.
This specimen was formalin-fixed, PMMA-embedded then processed for the cryofracture technique and reembedded in PMMA. The implant
occupied the top space in this figure (magnification x 131,200).

junctional epithelium cells a normal complement of

organdes including endoplasmic reticulum, Golgi and
numerous tonofilaments. The one type of organdie
was not expected in such great abundance was the
secretory vesicle. These vesicles were associated with

that

Golgi, were numerous throughout the cytoplasm of the

cell and were observed fused with the outer plasma
membrane suggesting a mechanism for discharge of

their vacuole contents. Presence of these vesicles around

the Golgi and throughout the cytoplasm further suggests that they were produced by the cells in concert
with current accepted cell biology doctrine, i.e., protein
and other materials are produced by the endoplasmic
reticulum and transported to the Golgi where the material is packaged in membrane-bound vesicles. The
vesicles then move through the cytoplasm to eventually

discharge

their product via exocytosis to the external

environment. This type of process would seem to account for the presence of the flocculent, dense material
on the outer cell membrane which is similar in density
to the material contained in the vesicles. Indeed, this
may partially explain how the basal lamina and other
extracellular proteoglycans on the external surface of
the junctional epithelial cells are produced. The reason
that there are several morphological populations of
vesicles is not clear. However, it is known that keratohyalin granules in oral mucosa, which are involved in
orthokeratin or parakeratin differentiation activities, do
have varying morphological features suggesting different functions for the granules.34 It may be that there
are specific targeted reasons for each of these vesicles,
one to produce the basal lamina, the other to produce

588

J. Periodontol.
October, 1985

McKinney, Stefiik, Roth

Figure 17. This epithelial cell exhibits numerous hemidesmosomes along the unit membrane with prominent peripheral densities (h). The
pyramidal particles and fine filaments are not as distinct as in Figures 15, 16 and 18. The lamina densa varies from an even outer surface (e) to
a jlocculent uneven (u) surface. Note the complex organization of the lamina densa at both e and u. This specimen was formalin-fixed, PMMAembedded then processedfor the cryofracture technique and reembedded in PMMA. The implant occupied the top space in this figure (magnification
110,700).

Figure 18. A junctionai epithelial cell unit membrane with long hemidesmosomes revealing their substructure ofperipheral densities and pyramidal
particles (below dots). The lamina lucida is narrower than previous figures. The complex organization of the lamina densa (Id) and the outer
dense linear body (lb) is revealed (see text). A sublamina lucida occurs al s. This specimen was formalin-fixed, PMMA-embedded then processed
for the cryofracture technique and reembedded in PMMA. The implant occupied the top space in this figure (magnification x 170,00).

additional extracellular matrix components such as glycosaminoglycans or proteoglycans. Further study is

needed to clarify this point. Despite the unclear nature
of the multiple vesicle population, the presence of a
large number of vesicles aggregated at the junctionai
epithelial surface very strongly supports the supposition
that the epithelial cells produce their own basal lamina
and other extracellular attachment components and
thus are directly responsible for their survival in the
oral environment.
Hemidesmosomes. The hemidesmosome organelle
was of great interest because it is intimately involved in
epithelial attachment to natural teeth30"32 and has been
observed ultrastructurally by other implant scientists.141517"19 We initially observed hemidesmosomes
on our microdissected specimens as dense, plaque-like
configurations on the outer unit membrane. Because

specimens lacked a well-structured basal

lamina, we were concerned that we might not be positively observing hemidesmosomes. When we employed
high resolution microscopy of the microdissected specimens, we were able to show the unit structure of the
hemidesmosomes complete with fine filaments running
into the cell cytoplasm (Figs. 4 and 6).
Upon using the alternative cryofracture technique,
we significantly improved our ability to preserve the
integrity and intimate detail of the external epithelial
surface interfacing the dental implant. Present were the
inner and outer peripheral densities, pyramidal particles
and fine filaments of the hemidesmosome (Figs. 15 and
16) as detailed by others.30 The fine filaments coursed
through the inner peripheral density from the cell sap
and extended through the outer peripheral density into
the lamina densa (Figs. 15 and 16).
these latter

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Basal Lamina. Early ultrastructural examination of

the microdissected specimens did not reveal a prominent basal lamina, only irregular flocculent densities
along the outer plasma membrane (Fig. 13). Instead of
accepting this as lack of evidence for a basal lamina
interfacing implants, we considered the possibility that
the microdissection technique had torn the extracellular
attachment structures or had created an artifact. Thus,
we employed the cryofracture technique. However, the
only specimens remaining that could be used for this
procedure were the formalin-fixed PMMA-embedded
specimens. Fortunately, the formalin fixation was adequate to preserve satisfactory ultrastructural detail and
the basal lamina, plasma membrane and hemidesmosomes were vividly displayed by this procedure (Figs.
14-18). Use of cryofracture not only revealed new
ultrastructural details, but also demonstrated the necessity of employing more than one procedure in undertaking morphological studies.
The basal lamina appeared as a definitively layered
structure on the outer junctional epithelial unit membrane. Based on the degree of resolution in the high
magnification transmission electron micrographs, the
substructures of the basal lamina, the lamina lucida
and the lamina densa were evident. A sublamina lucida,
as described by Kobayashi and co-workers,30 was fortuitously observed in some high resolution micrographs
(Fig. 18). The lamina densa had an uneven outer border
in some specimens and appeared to be composed of
two electron-dense complex structures separated by a
thin electron-lucent layer, the sublamina lucida (Fig.
18). The existence of this additional dense structure
outside the lamina densa could be analogous to the
linear border or dental cuticle as described by Kobayashi et al.30 Although the presence and function of
a dental cuticle is debated, the fact remains that these
dense structures have been ultrastructurally shown in
natural teeth.3032 The cuticle is theorized to be the
biological layer between the tooth surface and the sublamina lucida of the basal lamina. The linear border is
the dense structure observed between afibrillar cementum and the basal lamina in place of a dental cuticle.30
James and Kellen4 earlier suggested that a dental cuticle
did exist between implant and junctional epithelium
based on their histological periodic acid-Schiff (PAS)
staining studies. We do not agree with their observation,
or the concept that a dental cuticle exists around implant posts, but we do agree that there conceivably
exists a linear border structure outside the lamina densa
that is intimately involved with the attachment apparatus. The linear border is well defined and probably
represents the condensation of extracellular matrix proteoglycans involved in the attachment of the basal
lamina to surrounding structures. Since filaments or
protein structures cannot insert into inert biomaterials
as they do into cementum, the linear border in our
view is a very important structure that may represent
condensation of proteoglycans, possibly fibronectin,

Junctional Epithelial Attachment to Ceramic Implants 589

that attaches the lamina densa to a coating of glycosaminoglycans on the surface of the ceramic biomaterial.
The Attachment Complex. The ultimate question
remains as to what is the nature of the molecular
adhesion of the junctional epithelium to the implant.
Although this question is not completely answered in
this study, the ultrastructural data presented can be
combined with some current concepts of cell biology
to achieve a better understanding of the implant-tissueinterface attachment complex.
Laminin is an extracellular glycoprotein that is located in the lamina lucida and is probably responsible
for the adhesion of the epithelial cells to the collagenous
basal lamina.37 38 It is likely produced by the epithelial
cells themselves,39 and this would agree with our observations of the material contained in secretory vesicles
being similar in density to material attached to the
plasma membrane.
The lamina densa is devoid of laminin but has present another glycoprotein involved in adhesion activi-

ties, fibronectin, a glycoprotein produced by fibroblasts

and endothelial cells and found in the extracellular

matrix, especially during wound healing.40 Fibronectin
binds to collagen and glycosaminoglycans,41 and thus
may represent the "glue" between the implant biomaterial and the basal lamina's lamina densa which is
composed of Type IV collagen.42
The glycosaminoglycans, hyaluronic acid, heparin
sulfate and heparin (called proteoglycans when com-

plexed to a protein core), are produced by fibroblasts

during the healing phase following implant insertion
and may coat the implant surface. Fibronectin, likewise
produced by these same fibroblasts, is present in the
extracellular matrix. As the junctional epithelium regenerates, producing its own basal lamina and laminin,
the fibronectin provides the bond between the proteoglycans (or glycosaminoglycans) on the implant surface
and the lamina densa of the basal lamina. Thus, the
dense outer layer, or linear body of Kobayashi et al.,30
seen in our study may be the electron-dense staining of
the glycoprotein fibronectin and may portray the ulti-

biological seal.
The hemidesmosomes are undoubtedly epithelialspecific, adhesion plaques43 that "tack" the plasma
membrane to adjacent structures or substrate. Interestingly, the adhesion plaques, and presumably the hemidesmosomes, do not contain fibronectin,44 although
another actin-binding protein, vinculin, is present.43
Conclusions. This study, using ceramic alumina oxide endosteal implants, demonstrates the ultrastructural
presence of anatomical structures that represent the
biological attachment of gingival epithelium to the dental implant. Present are hemidesmosomes, basaflamina
and a linear border on the external unit membrane of
the epithelial cells. These structures are similar in nature
to those observed in mature31,32 and regenerated35 36
junctional epithelium adjacent to a tooth. The presence
of these structures provides tangible biological evidence
mate ultrastructural

590

i. Periodontol.
October. 1985

McKinney, Stefiik, Koth

for the presence of a per-perimucosal or biological seal2

at the tissue-implant interface and validates current
clinical studies demonstrating satisfactory patient service with ceramic implants.45'46
REFERENCES
1.

Lavelle, C. L. B.: Mucosal seal around endosseous dental im-

plants. J Oral Implantai 9: 357, 1981.

2. McKinney, R. V., Jr., Stefiik, D. E., and Koth, D. L.: Per, peri
or trans? A concept for improved dental terminology. J Proslhet Dent
52:267, 1984.
3. Klawitter, J. J., and Hulbert, S. F.: Application of

porous

ceramics for the attachment of load bearing internal orthopedic

applications. J Biomed Mater Res, Symposium No. 2(Part I). 161,
1971.
4. James. R. ., and Kellen, E. E.: A histopathological study of
the nature of the epithelium surrounding implant posts. Oral Implantla: 105 and 137, 1973.
5. Gross, U. M., and Strunz, V.: Surface staining of sawed sections
of undecalcified bone containing alloplastic implants. Satin Technol
52: 217, 1977.
6. Stefiik, D. E.. McKinney, R. V., Jr., Mobley, G., and Koth, D.
L.: Simultaneous histological preparation of bone, soft tissue and
implanted biomaterials for light microscopic observations. Stain
Technol 57: 91, 1982.
7. Bodine, R. L., and Mohammed, C. I.: Histologie studies of a
human mandible supporting an implant denture. J Prosthet Dent 21:

203, 1969.
8. Natiella, J. R., Armitage, J. E., Meenaghan. . ., and Greene,
G. W.: Tissue response to dental implants protruding through mucous
membrane. Oral Sci Rev 5: 85, 1974.
9. James, R. ., and Kellen, E. E.: A histological report on the
nature of the epithelium and underlying connective tissue which

implants. II. J Biomed Mater Res, Symposium No.

373,
1974.
5(Part 2),
10. Brunski. J. B., Moccia, A. F., Jr., Pollack, S. R., et al.: The
influence of functional use of endosseous dental implants on the
tissue-implant interface. I. Histological aspects. J Dent Res 58: 1953,
surrounds oral

1979.
11. McKinney, R. V., Jr., and Koth, D. L.: The single crystal
sapphire endosteal dental implant: material characteristics and 18month experimental animals trials. J Prosthet Dent 47: 69, 1982.
12. McKinney, R. V., Jr., Stefiik, D. E., and Koth, D. L.: The
biologic response to the single-crystal sapphire endosteal dental implant: scanning electron microscopic observations. J Prosthet Dent

51: 372, 1984.

13. McKinney, R. V., Jr. Stefiik, D. E., and Koth, D. L.: Ultrastructural surface topography of the single crystal sapphire endosseous
dental implant. J Oral Implantol 11: 327, 1984.
14. James, R. ., and Schultz, R. L.: Hemidesmosomes and the
adhesion of junctionai epithelial cells to metal implants. A preliminary report. J Oral Implantol 4: 294, 1974.
15. Listgarten, . ., and Lai, C. H.: Ultrastructure of the intact
interface between an endosseous epoxy resin dental implant and the
host tissues. J Biol Buccale 3: 13, 1975.
16. Gould, T. R. L., Brunette, D. M., and Westbury, L.: The
attachment mechanism of epithelial cells to titanium in vitro. J
Periodont Res 16: 611, 1981.
17. Karagianes, M. T., Westerman, R. E., Hamilton, A. I., et al.:
Investigation of long term performance of porous-metal dental implants in nonhuman primates. J Oral Implantol 10: 189, 1982.
18. Gould, T. R. L.. Westbury, L.. and Brunette, D. M.. Ultrastructural study of the attachment of human gingiva to titanium in
vivo. J Proslhet Dent 52: 418, 1984.
19. Swope, E. M., and James, R. .: A longitudinal study on

hemidesmosome formation at the dental implant-tissue interface. /

Oral Implantol 9: 412, 1981.
20. Kawahara, H.: Today and tomorrow of bioceramics in artificial organ. Jpn Art ifOrgan 6: 218, 1977.
21. Kawahara, H., and Hirabagashi, M.: Single crystal alumina
for dental implants and bone screws. J Biomed Mater Res 14: 597,
1980.
22. McKinney, R. V., Jr., Koth, D. L., and Stefiik, D. E.: The
single crystal-sapphire endosseous dental implant. I. Material characteristics and placement techniques. J Oral Implantol 10:487, 1982.
23. Koth, D. L., and McKinney, R. V., Jr.: The single crystal
sapphire endosteal dental implant. J. W. Clark (ed), Clinical Dentistry, Chapter 62, vol 5. Philadelphia, Harper and Row, 1984.
24. McKinney, R. V., Jr., Koth, D. L., and Stefiik, D. E.: The
single crystal sapphire dental implant. II. Two year results of clinical
animal trials. J Oral Implantol 10: 619, 1983.
25. Stefiik, D. E., McKinney, R. V., Jr., and Koth, D. L.: A
statistical analysis of the clinical response to the single-crystal sapphire
endosseous dental implant in dog jaws. J Dent Res 62: 1212, 1983.
26. Luft, J. H.: Improvements in epoxy resin embedding methods.
J Biophys Biochem Cytol 9: 409, 1961.
27. Stefiik, D. E., McKinney, R. V., Jr., Koth, D. L., and Singh,
B. B.: The biomaterial tissue-interface: a morphological study utilizing
conventional and alternative ultrastructural modalities. Scan Electron
Microsc/II: 547, 1984.
28. Linkow, L. I.: Statistical analysis of 173 implant patients. Oral
Implantol 4: 540, 1973.
29. Cranin, . ., Rabkin, M. F., and Garfinkel, L.: A statistical
evaluation of 952 endosteal implants in humans. J Am Dent Assoc
94: 315, 1977.
30. Kobayashi, K, Rose, G. G., and Mahan, C. J.: Ultrastructure
of the dento-epithelial junction. J Periodont Res 11: 313, 1976.
31. Listgarten, . .: Electron microscopic study of the gingivodental junction of man. Am JAnat 119: 147, 1966.
32. Schroeder, H. E.: Ultrastructure of the junctionai epithelium
of the human gingiva. Helv Odontol Acta 13: 65, 1969.
33. Yamaski, ., Nikai, H., Niilani, K, and Ijuhin, N: Ultrastructure of the junctionai epithelium of germfree rat gingiva. J Periodontol
50: 641, 1979.
34. Jessen, H.: Two types of keratohyalin granules. / Ultrastruct
Res 33: 95, 1970.
35. Taylor, A. C, and Campbell, M. M.: Reattachment of gingival
epithelium to the tooth. J Periodontol 43: 281, 1972.
36. Braga, A. M., and Squier, C. .: Ultrastructure of regenerating
junctionai epithelium in the monkey. J Periodontol 51: 386, 1980.
37. Terranova, V. P., Rohrbach, D. ., and Martin, G. R.: Role
of laminin in the attachment of PAM 212 (epithelial) cells to basement membrane collagen. Cell 22: 719, 1980.
38. Farquhar, M. G.: The glomerular basement membrane. E. D.
Hay (ed), Cell Biology of Extracellular Matrix, pp 335-378. New
York, Plenum, 1981.
39. Hogan, B. L. M., Cooper, A. R., and Kurkinen, M.: Incorporation into Reichert's membrane of laminin-like extracellular proteins
synthesized by parietal endoderm cells of the mouse embryo. Dev
Biol SO: 289, 1980.
40. Hakomori, S., Fukuda, M., Sekiguchi, K., and Carter, W. G.:
Fibronectin, laminin, and other extracellular glycoproteins. K. A.
Piez and A. H. Reddi (eds). Extracellular Matrix Biochemistry, pp
229-275. New York, Elsevier, 1984.
41. Oldberg, ., and Ruoslahti, E.: Interactions between chondroitin sulfate proteoglycan, fibronectin, and collagen. J Biol Chem 257:
4859, 1982.
42. Timpl, R., Wiedemann, H., van Delden, V., et al.: A network
model for the organization of type IV collagen molecules in basement
membranes. Eur J Biochem 120: 203, 1981.
43. Geiger, .: A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken
cells. Cell 18: 193, 1979.

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Number 10

44. Chen, W. T., and Singer, S. J.: Fibronectin is not present in

the focal adhesions formed between normal cultured fibroblasts and
their substrata. Proc Nati Acad Sci USA 77: 7318, 1980.
45. Koth, D. L., McKinney, R. V., Jr., and Davis, Q.: The singlecrystal sapphire endosteal dental implant. A longitudinal human
study, one year results. J Prosthet Dent 50: 72, 1983.
46. Stefiik, D. E., Koth, D. L., and McKinney, R. V., Jr.: A

Junctional Epithelial Attachment to Ceramic Implants 591

clinical and statistical analysis of human clinical trials with the single
crystal sapphire endosteal dental implant: two year results. J Oral
Implanto! 11: 500, 1984.
Send reprint requests to: Dr. Ralph V. McKinney, Jr., Department
of Oral Pathology, School of Dentistry, Medical College of Georgia,
Augusta, GA 30912.