Journal of Periodontology 1989 Apr (188 - 198): Cell and Fiber Attachment to

Demineralized Cementum From Normal Root Surfaces P. J. Hanes, A.M. Polson

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Cell and Fiber Attachment to Demineralized Cementum From Normal Root
Surfaces
P. J. Hanes and A. M. Polson

THE PURPOSE OF THIS STUDY was to assess connective tissue and epithelial
responses to cementum (from normal human root surfaces covered by periodontal
ligament) after surface demineralization with citric acid. Each rectangular specimen had
a face of cementum and an opposite surface composed of pulpal dentin. One half of the
specimens were treated with citric acid (experimental group), while the remainder
served as untreated control specimens. Specimens were implanted vertically into
incisional wounds on the dorsal surface of rats with one end of the implant protruding
through the skin. Four specimens in each group were available for examination 1, 3, 5,
and 10 days after implantation. Histologic and histometric analyses of the implants
included counts of adhering cells, evaluation of attached connective tissue fiber density
and diameter, and assessment of epithelial migration. At day 1, a distinct lighter staining
zone was present on the surface of both cementum and dentin in the experimental group
which corresponded to a zone of surface demineralization produced by the acid
treatment. Histometric comparisons between experimental and control groups at 10 days
showed a greater number of cells attached to demineralized cementum surfaces. Also, a
connective tissue fiber attachment system had developed on these experimental
surfaces, but which differed morphologically from periodontal ligament fiber
attachment to normal cementum. It was concluded that citric acid treatment can surface
demineralize cementum from normal roots, and that the surface demineralization of this
cementum facilitated a cell and fiber attachment to the cementum surface.

Surface demineralization of root surface with citric acid has been shown to predispose
toward connective tissue attachment with inhibition of epithelial migration.1-3 The
latter results have been obtained when the demineralized root surface has been first root
planed in order to remove accretions and cementum. However, there has also been lack
of predictability with this technique.4-7 It has been suggested that this inconsistency
may be due to variables such as inadequate removal of root surface contaminants,8-12
improper adaptation of the connective tissue flap to the root surface,2,13-15 and failure
to remove the cementum prior to application of the demineralizing solution.11
New connective tissue attachment to demineralized root surfaces has been reported to
occur in various animal models,2,13-15 and in humans.1,16 The regeneration reported
in these latter studies occurred in environments containing periodontal progenitor cells,
and these cells are thought to be necessary for the regeneration of new cementum,
periodontal- ligament, and alveolar bone.17-19 More recent studies, however, have
shown that connective tissue from non-periodontal origin can also be induced to attach
to a root surface which provides a biologically hospitable substrate.3,20-22 The root
surface which facilitated the connective tissue attachment in these studies was a dentin
surface, demineralized by citric acid. The demineralization of radicular dentin with
citric acid results in the exposure of the collagenous matrix of dentin,11,14,23 and this
substrate enhances the chemotaxis, migration, and attachment of connective tissue
cells.24-27 However, previous studies have reported conflicting results regarding the
exposure of collagen when cementum is treated with citric acid 11,25,26
Recent studies from our laboratories have shown that surface demineralization of dentin
with citric acid results in an enhanced cell and fiber attachment to the dentin, regardless
of whether it originated from either normal or periodontitis-affected root
surfaces.3,20,21 In light of these findings, it was the purpose of the present study to
evaluate the effects of citric acid treatment on the development of a cell and fiber
attachment system to cementum surfaces obtained from normal (i.e., non-periodontitis-
affected) roots. The method for evaluating in vivo epithelial and connective tissue
responses utilized a previously described implantation model system.3,20
MATERIALS AND METHODS
Preparation of Implant Specimens
Specimens for implantation were derived from non-periodontitis affected, unexposed
human root surfaces. Teeth with remnants of periodontal ligament fibers on their root
surfaces were selected from a pool of extracted human teeth. Following extraction, the
teeth had been rinsed in tap water and stored in water-moistened gauze at 30 to 40°F.
In order to assure the non-periodontitis affected status of these implants, specimens
were taken from the mid-root region, well apical to the coronal extent of periodontal
ligament fibers using a previously described method.3,21,22 (Fig. 1). Remnants of
periodontal ligament fibers were removed from the root surface with 12 strokes of a
sharp Gracey ½ curette. The remaining cementum on the root surface was not removed.
Thus, the root surface aspect was covered by a layer of cementum which had been
beneath attached periodontal ligament fibers. Each implant specimen was rectangular in
shape, approximately 3 mm long apico-coronally, 1 mm wide, and 1 mm thick. The
specimens were stored at 30 to 40°F in vials of tap water until used in experimental
procedures.
Experimental Procedures
The experimental animals consisted of 13 Sprague Dawley CD rats, approximately 180
days old and weighing 500 to 600 grams, which were maintained on an ad libitum diet
of Purina Rodent Chow and water. The animals were sedated for experimental
procedures with a combination of Ketaset (Ketamine Hydrochloride, 100 mg/ml) 50
mg/kg body weight and Rompun (Xylazine, 20 mg/ml) 5 mg/kg body weight.
The prepared specimens were implanted into the back of the rat's neck, between its
shoulder blades and skull.3,20 In each animal, immediately prior to implantation, two
specimens (experimental) were immersed in a solution of citric acid, pH = 1, for 3
minutes.2,3,11 After acid immersion, the specimens were rinsed in water and implanted
into the incisional wounds on the right side of the animal. Specimens were implanted,
with long axis vertical, so that approximately 1 mm of the implant protruded above the
skin. The specimens implanted on the left side of the animal served as nonacid treated
controls. No form of stabilization was used to secure the specimens.
Animals were sacrificed in carbon dioxide derived from dry ice at 1, 3, 5, and 10 days
following placement of implants.3,20 Four experimental and four control specimens
were available at each time point. Biopsy methods and preparation of specimens for
histologic analysis were as described in previous studies.3,20 Serial histologic sections
were cut with the microtome set at 6 mm and sections representing intervals of 120 mm
were stained with hematoxylin and eosin. Sections preceding those stained with
hematoxylin and eosin were stained with a silver impregnation technique to delineate
connective tissue fibers.
Methods of Analysis
A histologic and histometric analysis was done using three step-serial sections from
each specimen. Sections for analysis consisted of the mid-section and one step-serial
section on either side of the mid-section. The epithelium and connective tissue adjacent
to the root surface and pulpal surface of the implant were examined.
The histometric analyses consisted of measuring the following parameters:
1. Length of cementum surface within connective tissue: This dimension was measured
at a magnification ´ 40 in an Olympus BH2 microscope. The microscope was equipped
with a digitizing system attached to an Apple II+ computer. Using computer software
for histometric analysis, the length measured was the distance between the apical
termination of the epithelium on the cementum and the apex of the cementum surface.
2. Number of attached cells: The number of connective tissue cells attached to the
cementum surface of the implant was counted using a light microscope and
magnification of ´600. Cells were counted from the apical extent of the epithelium to
the apical end of the implant. A cell was counted as attached if its nucleus either (1)
contacted the implant surface or, (2) was adjacent to the surface and lay within
intercellular matrix which was attached to the implant surface. Nuclear fragments were
counted as one cell. The number of attached cells was expressed per 100 mm of surface
length.
3. Density of attached fibers: The number of fibers attached per 100 mm of cementum
surface (root aspect) or dentin surface (pulpal aspect) was counted in the 10-day
specimens using the sections stained with the silver impregnation technique at
magnification ´600. A fiber was classified as attached if it was continuous with the
implant surface.
4. Diameter of attached fibers: The diameter of fibers attached to 10-day specimens was
measured at a magnification of ´ 1000 using the Olympus microscope and the digitizing
pad. The digitizing system was used to measure the diameter of ten randomly selected
attached fibers on both the root and pulpal surfaces of the 10-day specimens. The
measurement of fiber diameter was made as close as possible to the implant surface. In
addition, the diameter of ten connective tissue fibers present in the animal's loose
connective tissue surrounding the implant was measured on each of the histological
sections taken from the 10-day experimental group. A mean fiber diameter was
calculated for each category (root cementum surface, pulpal dentin surface, loose
connective tissue) from the component fiber measurements.
Data Management
Values from the individual sections of the experimental and control specimens were
used to calculate a mean for each measured value for the surface of the specimen. An
overall mean, for the time point was calculated from the separate specimen means.
Statistical comparisons using Student's t-test were made between cementum surface
means in the experimental and control groups at each time point. The number of cells
attached to cementum surfaces within experimental and control groups were compared
across time using analysis of variance. In the event of a significant overall analysis of
variance, a Mann-Whitney U test was used to assess the significance of differences
among and between the time points. Statistical comparisons between fiber data obtained
from implant specimens and the loose connective tissue were also made using Student's
t-test. A level of P< 0.05 was accepted for statistical significance.
RESULTS
Histological examination of control specimens at 1 day showed the vertical implant in
place within the connective tissue and protruding through the surface epithelium (Fig.
2). It was possible to distinguish the pulpal dentin surface with the notch from the root
surface aspect which was covered by cementum. The epithelium on the surface of the
skin terminated at the wound margin adjacent to the cementum surface (Fig. 3).
At high magnification on the cementum surface, there were cells attached to the
cementum or enmeshed within a fibrin network adjacent to the implant surface (Fig. 4).
The majority of cells appeared to be inflammatory cells. In specimens examined 3 days
after implantation, cells remained attached to the cementum surface (Fig. 5). The
epithelium was migrating apically over the connective tissue adjacent to the cementum
surface (Fig. 6).
In specimens examined 5 days after implantation, apical migration of the epithelium had
progressed and extended virtually to the apex of the specimen (Fig. 7). Inflammatory
cell populations in the connective tissue adjacent to the implant surfaces were reduced,
and a more collagenous connective tissue was present. There did not appear to be any
connective tissue fiber attachment to the implant surfaces.
Various degrees of implant extrusion and exfoliation were present in specimens
examined at 10 days after implantation (Fig. 8). The length of the implant surface within
the connective tissue was significantly less than had been present at the 1 day time point
(Table 1). In most specimens, epithelium had migrated to the base of the implant. In
areas where connective tissue remained adjacent to the implant surface, evidence of
fiber attachment to the implant was sparse (Fig. 9). The implants did not show evidence
of resorption.
Experimental specimens, at day 1 after implantation, were vertically located within the
connective tissue with one end protruding through the surface epithelium at the margin
of the wound (Fig. 10). The length of the cementum surface within the connective tissue
did not differ significantly between experimental and control group specimens at the 1
day time point (Table 1). A cell-rich fibrin network was adjacent to the root surface
aspect of the implant, and many cells appeared to be attached to the cementum (Fig. 11).
The cementum had a lighter staining surface zone, approximately 12 mm in width.
At 3 and 5 days, the connective tissue was apposed against the root surface, and many
cells were still attached (Fig. 12). The specimens at 10 days showed an intimate
apposition of the connective tissue to the implant surfaces (Fig. 13). Apical to the
epithelium, there was a cellular connective tissue (Fig. 14), which had fiber attachment
to the implant surface (Figs. 15, 16). The attached fibers were oriented obliquely and
perpendicular to the implant surface. A significantly greater number of fibers were
attached to demineralized cementum compared to dentin from the pulpal surface (Fig.
17, Table 2). In addition, those fibers attached to the demineralized cementum were
larger in diameter than fibers attached to demineralized pulpal dentin (Table 2).
Although those fibers attached to demineralized pulpal dentin were similar in diameter
to connective tissue fibers present in the loose connective tissue, those fibers attached to
the demineralized cementum surfaces were significantly greater in diameter than those
within the loose connective tissue (Table 3).
The histometric analysis of counts of cells attached to the cementum surfaces showed a
tendency for a greater number of cells to be attached to demineralized cementum at each
time point. However, the differences were not statistically significant until 5 and 10
days after implantation (Table 4). In the control group, the number of cells attached to
the cementum surface decreased significantly with time (Table 5). Although a similar
trend was observed in the experimental groups, the differences across time were
inconsistent.
DISCUSSION
The purpose of this study was to evaluate cellular, connective tissue, and epithelial
responses to cementum surfaces which had been obtained from the roots of normal
human teeth and subsequently treated with citric acid. An animal model was utilized
which enabled the evaluation of healing in an environment lacking periodontal
progenitor cells. Based on the findings of this study, it was concluded that there were
marked differences in the in vivo connective tissue response between the experimental
(citric acid treated) and control (non-citric acid treated) groups. Surface
demineralization of cementum from normal roots predisposed toward the development
of a cell and fiber attachment system. In addition, this cell and fiber attachment occurred
in an environment lacking periodontal progenitor cells, thereby emphasizing the
importance of root surface characteristics.
Citric acid treatment appeared to demineralize the cementum surface and expose the
collagenous matrix of the cementum to a depth of approximately 12 mm. This depth of
surface demineralization is greater than the 3 to 5 mm depths reported by previous
studies following citric acid treatment of dentin,2,11,14 and may be related to the higher
ratio of organic to inorganic components that is present in cementum.28,29 In a
previous study,11 citric acid treatment produced no apparent alteration in root surface
morphology unless the root was first root-planed in order to remove the cementum. The
conflicting results of the present study, however, may be related to the different origins
of the cementum surfaces in the two studies. In our present study, cementum surfaces
were derived from areas beneath attached periodontal ligament fibers on unexposed,
non-periodontitis affected root surfaces. On the other hand, the cementum surfaces in
the previous study11 were obtained from exposed, periodontitis-affected root surfaces.
Earlier studies have shown that exposed cementum may be high in fluoride content,30or
hypermineralized as a consequence of periodontal disease,31,32 and may inhibit the
effectiveness of the acid demineralization .
There were a greater number of cells attached to demineralized cementum surfaces at all
time points following implantation. However, the differences between cell attachment to
demineralized and non-demineralized cementum surfaces were not significant until 5
and 10 days following implantation (Table 4). At earlier time points, there was a
tendency for greater cell attachment to non-demineralized cementum surfaces than has
been reported for non-demineralized dentin surfaces in this model system.3,20
However, the number of cells attached to cementum surfaces in the present study
decreased with time (Table 5) in a manner similar to that reported previously for
dentin.3,20 This tendency for a greater cell attachment to non-demineralized cementum
may be due to the higher collagen content of cementum.28,29 Type I collagen and its
degradation products are known to be chemotactic stimulants for PMNs, macrophages,
and fibroblasts.24,33,34 In addition, more recent studies have demonstrated that non-
collagenous extracts of cementum have the ability to stimulate the attachment,
spreading and DNA synthesis of gingival and skin fibroblasts.35-39 It is conceivable,
therefore, that at early time points, factors present in the cementum, including collagen
and human cementum growth factor,35,37 may enhance the chemotaxis, migration, and
attachment of cells on both demineralized and non-demineralized cementum surfaces.
In this respect, it should also be recalled that the control cementum surfaces were
prepared by curetting a normal root surface with attached periodontal ligament fibers.
This type of instrumentation results in the formation of a covering "smear" layer.40-42
If this occurred in our investigation, the composition of the layer may well have
contained many collagenous and cemental elements conducive for initial cell migration
and attachment.
The greater cell attachment associated with demineralized cementum surfaces at later
time points (Tables 4, 5), may well be related to the increased exposure to the collagen
substrate of cementum by the citric acid treatment. In view of the fact that fibroblasts
increase in number as healing progresses,43-46 fibroblasts would likely comprise a
greater proportion of those cells attached to cementum at later time points. As fibroblast
adhesion and locomotion is facilitated by collagen,45-47 a preferential attachment of
fibroblasts to a collagen substrate produced by the demineralization of cementum could
result-in the elaboration of glycosaminoglycan ground substance45 and collagen
fibrils46 at the root surface interface. Such events would be consistent with the
formation of a connective tissue attachment to a root surface. These findings regarding
initial cell attachment and later presence or absence of fiber attachment underscore that
it may not be sufficient to have increased numbers of cells present, but they must be
stimulated and activated to produce the correct biological sequence of events for
subsequent fiber attachment 3,27,47
A distinct connective tissue fiber attachment occurred to demineralized cementum
surfaces between 5 and 10 days after implantation. The development of this fiber
attachment occurred during the time in which fibroblast populations peak and collagen
content increases in the wound healing process, namely, 5 to 10 days post injury.46
Both the number of attached fibers per unit length, and the diameter of attached fibers
was significantly greater on cementum than dentin surfaces (Table 2).
The density and diameter of the fibers attached to the demineralized pulpal dentin
surfaces was similar to those forming on radicular dentin surfaces reported previously in
this model system.3,20,22 The diameter of fibers attached to the pulpal dentin surfaces
was considerably less than that reported for the single periodontal ligament or Sharpey's
fiber bundle in the periodontium.3,48,49 It was similar, however, to the diameter of
collagen fibers present in the animal's loose connective tissue (Table 3). Thus, the
morphology of this cell and fiber attachment system which occurred to the dentin had
characteristics intrinsic to the connective tissue location.3,20,22
It was very interesting that both the density and diameter of the fiber attachment system
which developed on the demineralized cementum surfaces were significantly greater
than those associated with the pulpal dentin (Table 2). However, as the fibers which
attached to cementum surfaces were followed into the adjacent connective tissue, their
diameter decreased, tapering to a fiber of a diameter similar to that of adjacent loose
connective tissue fibers (Figs. 15, 16). It has been suggested that connective tissue
attachment to citric acid-treated root surfaces occurs by means of a "splice" between the
collagen fibers in the connective tissue, and the collagen fibrils exposed on the root
surface by citric acid demineralization.14,15 Such a phenomenon could explain the
morphology of the fiber attachment to cementum surfaces in the present study. Citric
acid demineralization may expose the aggregates of collagen fibers which are embedded
in the cementum as a series of dome-shaped mounds.50-52 These larger fiber
aggregates could have acted as a "morphological template," and subsequently been
"spliced" to collagen fibers of smaller diameter present in the adjacent connective
tissue. This splicing could be facilitated by the elaboration of glycosaminoglycans and
collagen fibrils by fibroblasts attached to the cementum surface.45,46
The morphology of the fiber attachment system which occurred to demineralized
cementum surfaces in the present study differed in several respects from the periodontal
ligament fiber system which provides the attachment to cementum in a normal
periodontium. Firstly, the number of attached fibers per unit length was less than that
reported for the normal periodontal ligament.3 In addition, the diameter of those fibers
attached to cementum surfaces in the present study was considerably less than that
reported for the diameter of fibers of the periodontal ligament.49-52 Although the
morphology of the fiber attachment system which occurred to demineralized cementum
surfaces differed substantially from that of the normal periodontal ligament fiber
attachment, the presence of morphological, oriented fiber continuity between the
cementum surfaces and the adjacent soft connective tissue suggested that connective
tissue can be induced to attach to a cementum surface having appropriate substrate
characteristics. 26,27,42,53
In summary, the present study evaluated cellular, connective tissue, and epithelial
responses to cementum surfaces which had been obtained from the roots of normal
human teeth, and subsequently demineralized with citric acid. The results showed that
citric acid treatment can expose the collagenous matrix of cementum. Furthermore,
surface demineralization of cementum predisposed toward the development of a cell
and fiber attachment system. This cell and fiber attachment system occurred in an
environment lacking periodontal progenitor cells, thereby emphasizing the importance
of cementum surface characteristics. The morphology of the cell and fiber attachment
system differed substantially from the normal attachment of periodontal ligament to the
human root surface, and had characteristics related to the surface morphology of the
cementum and the connective tissue location. It would be of interest to use this model
system to test the ability of citric acid to surface-demineralize periodontitis-affected
cementum, and evaluate the potential for new connective tissue attachment to these
treated cementum surfaces.
ACKNOWLEDGMENTS
Study supported in part by Grant Number DE07061 from the National Institute of
Dental Research, ,National Institutes of Health, Bethesda, MD, and the Pluta
Periodontal Fund.
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