In Vitro Growth of Human Gingival Fibroblasts on Root

Surfaces of Endodontically Treated Teeth* f

Robert M. Dunlap4 Jonathan L. Gray,§ Donald W. Turner,|| Arthur
R. Vernino,If John E. Williams, Jr.,** and George B. Pelleu, Jr.ft

Controversy the potential for new connective tissue attachment to dentin of

exists over
endodontically-obturated teeth following periodontal therapy. The purpose of this study was
to determine whether cultured human gingival fibroblasts would grow in vitro on planed
dentin surfaces of endodontically-treated teeth. A model similar to that of Aleo et al. was
developed using extracted endodontically-treated human teeth. This model consisted of
longitudinally sectioning 10 teeth, root planing one section only, and incubating both sections
with a suspension of human gingival fibroblasts. Fibroblast growth was detcimined by staining
with neutral red and trypan blue. The criterion for growth was staining of the complete root
surface. All root areas with attached periodontal fibers displayed staining, as well as the
complete root surface of all planed sections. Unplaned sections did not stain on root areas
formerly exposed to the oral environment owing to periodontal disease, a finding consistent
with the results of Aleo et al. Our results indicate that root canal therapy does not interfere
with in vitro growth of fibroblasts on planed dentin surfaces of endodontically-treated teeth.
Extrapolation to a clinical situation would indicate that normal healing may be expected after
periodontal surgery on tissues adjacent to root planed endodontically-treated teeth.

Controversy exists as to the potential for regeneration
of an attachment apparatus to the dentinal surface of an
endodontically-obturated tooth. There is evidence that
regeneration is possible to cementum1'2 and dentin1"6 of
teeth with vital pulps. New attachment to the cementum
of teeth that had nonvital pulps has also been docu-
mented.7 However, few authors have considered the new
attachment potential at the root-soft tissue interface on
the endodontically-treated tooth, and their findings dis-
agree. Morris,7'8 using human teeth with nonvital pulps,
* This
project was supported through funds provided by the Bureau
of Medicine and Surgery under Research Work Unit No. M0095.003-
3014.
f The opinions and assertions contained herein are the private ones"
of the writers and are not to be construed as official or as reflecting the
views of the Department of the Navy.
Commander, DC, USN; Periodontist, Branch Dental Clinic, U.S.
Naval Academy, Annapolis, MD; formerly, Resident in Periodontics,
National Naval Dental Center, Bethesda, MD.
§ Commander, DC, USN; Periodontist, Branch Dental Clinic,
Quantico, VA; formerly, Resident in Periodontics, National Naval
Dental Center.
II Captain, DC, USN; Deputy Chairman, Dental Sciences Depart-
ment, and Head, Laboratories Division, Naval Medical Research
Institute, Bethesda.
Captain, DC, USN; Chairman, Periodontics Department, Na-
tional Naval Dental Center.
**
Captain, DC, USN (Ret.); Periodontics Department, Indiana
University School of Dentistry, Indianapolis, IN; formerly, Chairman,
Periodontics Department, National Naval Dental Center.
ft Chairman, Research Department, National Naval Dental Center.
140
was unable to demonstrate cementogenesis on dentin of
endodontically-obturated teeth. There are clinicians and
researchers, on the other hand, who believe that new
attachment can be achieved on dentin of endodontically-
obturated teeth. The work of Bjorn,9,10 Diem,11 and
Mitsis12 indicates that endodontically-obturated teeth
should respond to periodontal therapy as do vital teeth.
Aleo et al.,13 in a recent study on extracted teeth,
investigated the biological compatibility between the
human gingival fibroblast and the root surface of peri-
odontally-involved teeth. Focusing on the segment of the
root surface which was previously exposed to the oral
environment, they showed that this segment was not
compatible with fibroblast growth. It was suggested that
the exposed cementum was contaminated with bacterial
endotoxin. Root surfaces treated with phenol in H20 or
root planing were found biologically acceptable for fi-
broblast growth. However, these investigators evaluated
fibroblast growth only on vital teeth, and the controversy
of new attachment regeneration on endodontically-ob-
turated teeth still exists. The present study was under-
taken, utilizing Aleo's tissue culture method, to deter-
mine if human gingival fibroblasts would grow on the
planed root surfaces of extracted endodontically-obtur-
ated teeth.
MATERIALS AND METHODS
Ten periodontally-involved human teeth, which had
Volume 52
Number 3 Endodontically Treated Teeth 141

been endodontically obturated at least 1 year prior to

extraction, used.
were Nine of these teeth had been filled
with gutta-percha and one tooth with a silver point. Ten
periodontally-involved human teeth with vital pulps
prior to extraction served as controls.
After the crowns were removed, the roots were sec-
tioned longitudinally with a high-speed handpiece and
water coolant. The cementum on one section was re-
moved with a curette, while the other section was left
intact. Both sections were sterilized in an autoclave for
15 minutes. After sterilization, both sections of each root
were placed in the bottom of a single, sterile 3.5- X 8-
mm Petri dish with the external surface of the root facing

upward. A suspension of human gingival fibroblasts

containing 1 X 10s cells per milliliter, propagated prior
to use according to the method of Aleo,13 was added to
each Petri dish so that the fluid level of the suspension Figure 1. Two root sections of a previously vital tooth. The section on the
right was cultured with fibroblasts subsequent to root planing. Stained
totally immersed the root sections. The medium used for fibroblasts can be seen on the entire length of the section. The section on
propagating and culturing the cells with the root sections the left is the unplaned section. After the culture period, no stained
was Dulbecco's modified Eagle medium supplemented fibroblasts are visible on that segment of the root (P) previously exposed
with 10% fetal calf serum, 100 units of penicillin per ml, in the oral cavity as a result ofperiodontal disease.
0.1 mg of streptomycin per ml, and glutamine, 2 mM.
The root sections were incubated with cells for 72 hours
at 37°C, in 5% CO2 in air, in a humidified atmosphere.
Following incubation, the sections were washed with
distilled water, stained with 0.01% neutral red dye to
verify cell viability, fixed with ethanol-ether, and then
stained with 0.5% trypan blue for gross visualization of
cell growth. The segment of each section which was
previously exposed to the oral environment was graded
for growth or no growth and photographed for docu-
mentation.

RESULTS
The presence of fibroblast growth on root sections of
vital and endodontically-obturated teeth is shown in
Table 1. Growth was observed on all 10 root-planed
sections of the endodontically-obturated specimens. No
growth was observed on those segments of unplaned root
sections which were formerly exposed to the oral cavity.
No difference in fibroblast growth was detected between
the planed root sections of the vital (Fig. 1) and endo-
dontically-treated teeth (Fig. 2).
Table 1
Incidence of Fibroblast Growth on the Planed and Unplaned
Periodontally-involved Root Surfaces of Endodontically-Obturated and
Previously Vital Teeth*
Number of sections
Pulpal status -
Figure 2. Two root sections of an endodontically-obturated tooth. The
section on the left was cultured with fibroblasts subsequent to root planing.
Stained fibroblasts can be seen on the entire length of the section. The
section on the right is the unplaned section. After the culture period, no
stained fibroblasts are visible on that segment of the root (P) previously
exposed in the oral cavity as a result ofperiodontal disease.
DISCUSSION
The results obtained this tissue culture model
using
system indicate that the planed root surfaces of the
endodontically-obturated teeth were compatible with fi-
broblast growth. No difference in growth was noted on
the root-planed surfaces of vital teeth as compared to the

root-planed endodontically-obturated teeth.

Growth No growth As stated earlier, Morris7 showed that cementogenesis
Vital teeth
0
did not occur on dentin of endodontically-obturated
Unplaned 10
teeth. He suggested that this lack of cementogenesis
Root-planed 10 0
Endodontically-obturated teeth might be related to a lack of fluid exchange, an absence
Unplaned 0 10 of some inductive principle, or to a toxicity produced by
Root-planed 10 0 the root canal medications. Prichard,14 citing Morris's
*
That portion of the root formerly exposed to the oral environment. findings, suggested an altered treatment sequence in

142 Dunlap, Gray, Turner, Vernino, Williams, Pelleu
attachment regeneration procedures to teeth which re-
quire endodontic therapy. He recommended debride-
ment and medication of the canals but a delay in obtur-
ation until several weeks after intrabony surgery. Fur-
thermore, Simring15 postulated that the intensity and
rate of extension of marginal Periodontitis were aggra-
vated by prior endodontic therapy. In a recent report
investigating ffeeze-dried osseous allografts,16 a less fa-
vorable response was noted adjacent to the endodonti-
cally-obturated tooth in the treatment of intrabony de-
fects.
Our findings are at variance with those of investigators
who imply that teeth treated endodontically acquire
biologically altered root surfaces.
In clinical periodontal practice, it is not unusual to
encounter periodontally-involved endodontically-obtur-
ated teeth. When the root surface is exposed to peri-
odontal disease, changes in the cementum occur,17"20 and
apparently adequate root planning is essential for favor-
able results. The interpretation of the present study,
along with the work of Bjorn,9'10 Diem,11 and Mitsis,12
indicates that endodontically-obturated teeth should re-
spond to new attachment procedures as do teeth with
vital pulps. The possibility of adverse effects on wound
healing does not appear to be a valid justification for
delaying completion of root canal therapy until after
periodontal surgery.
ACKNOWLEDGMENTS
The authors thank Dr. Bruce Baum for providing the human
gingival fibroblasts from the Clinical Sciences Division, National In-
stitute of Dental Research, National Institutes of Health, Bethesda,
MD. Also, the technical assistance of Mr. Vincent Berzinskas is greatly
appreciated. We also wish to thank Ms. Shirley Starnes for assistance
in preparation of the manuscript.
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Send reprint requests to: Dr. George B. Pelleu, Jr., Chairman,
Research Department (Code 43), National Naval Dental Center, Be-
thesda, MD 20014.