434

The Effect of Chlorhexidine Treatment

of Root Surfaces on the Attachment of
Human Gingival Fibroblasts In Vitro
Charles D. Alleyn, * Robert B. O'Neal, * Scott L. Strong, * Michael J. Scheidt DDS, *
Thomas E. Van Dyke,f and James C. McPherson*

Chlorhexidine mouthrinse is a widely used adjunct in periodontal therapy due to

its bactericidal effects. The effect of this agent on chronic gingivitis and wound healing
following surgical therapy in animals and humans has been favorable. The re-establish-
ment of lost connective tissue attachment to the root surface following periodontal therapy
is a desirable goal in which the ability of periodontal ligament fibroblasts to reattach to
root surfaces of periodontally involved teeth is a critical event. Understanding the effect
of Chlorhexidine on fibroblast attachment will provide the rationale for its use during the
healing phase of periodontal surgery. For this study, impacted third molars were sectioned
into 4 pieces. Groups of 10 root pieces were exposed to 0.12% Chlorhexidine or saline
for 3 minutes followed by a distilled water rinse. The root pieces were incubated with
human gingival fibroblasts (HGF) using standard tissue culture techniques for 1, 2, 4,
6, and 8 hours. HGF were prelabeled with 3H-thymidine to a standard specific activity.
The surface area of each root piece was determined and the attached cells quantified by
using scintillation spectroscopy. The number of cells per unit area was then calculated
and the data expressed as cells/mm2. The repeated measures design was statistically
analyzed by repeated measures analysis of variance. There was a significant difference
between the number of attached cells in the Chlorhexidine and the control groups (P <
0.001). Exposure of root surfaces to Chlorhexidine significantly inhibits subsequent fi-
broblast attachment which may interfere with regeneration of the periodontium. Hence,
the data suggest that efforts should be made to minimize Chlorhexidine contact with the
root surface with physical barriers. / Periodontol 1991; 62:434-438.

Key Words: Periodontal diseases/surgery; chlorhexidine/therapeutic use; wound healing;

connective tissue; gingivitis/drug therapy; fibroblasts.

Chlorhexidine mouthrinse is a widely used adjunct in peri- that in dogs treatment with Chlorhexidine resulted in reso-
odontal therapy due to its bactericidal effect. Chlorhexidine lution of gingivitis and significantly lowered plaque scores.
is a cationic molecule and belongs to the polybiguanide Bogle et al.4 demonstrated greater bone, regeneration in bi-
group of compounds. The bactericidal effect of the drug furcation defects in dogs when topical Chlorhexidine was
results from the cationic molecule altering the osmotic equi- used post operatively. Langenbaek and Bay5 showed a re-
librium of the microbes.1 The effectiveness of Chlorhexi- duction of plaque index and gingival exúdate when a Chlor-
dine rinse as a plaque control agent is due largely to its hexidine mouthrinse was used after gingivectomy. A study
substantivity. It adsorbs on oral surfaces and then is slowly by Bakaeen and Strahan6 suggests that there may be less
released in active form.2 Numerous studies suggest the use postoperative pain when Chlorhexidine is used after peri-
of Chlorhexidine mouthrinse has a favorable effect on plaque odontal surgery, although they were unable to show any
control and gingival inflammation. Lindhe et al.3 showed significant differences in Plaque Index, Gingival Index,
crevicular fluid, or depth of pockets. Newman and Addy7
•U.S. Army, Ft. Gordon, GA. were able to demonstrate a reduction in plaque scores and
tCurrently, Eastman Dental Center, Rochester, NY; previously, Emory sulcus bleeding in patients who used Chlorhexidine post-
University, Atlanta, GA. operatively, but found no significant reduction of postop-
The opinions expressed in this article do not represent the views of the
United States Department of Defense, the Department of Army, or the
erative pain. A study by Asboe-Jorgensen et al.8 revealed
United States Dental Corps. Use of any commercial products in this project a reduction in gingival exúdate and a decreased tendency
does not imply endorsement by the U.S. Government. to bleeding following postoperative use of Chlorhexidine.
Volume 62
Number 7 ALLEYN, O'NEAL, STRONG, SCHEIDT, VAN DYKE, McPHERSQN 435

In contrast to the above, several studies provide evidence during extraction, were used. After removing any debris on
demonstrating toxic effects of Chlorhexidine on human cells the root surface with a sealer, the crowns were removed
and granulation tissue. Paunio et al.9 and Bassetti and and the cementum removed with a diamond bur. Removal
Tallenberger10 demonstrated that Chlorhexidine delays gran- of cementum was confirmed visually using a stereomicro-
ulation tissue formation. Cytotoxicity has been demon- scope. The roots were then sectioned into dentin chips which
strated in neutrophils.11,12 These studies were able to were rectangular in shape and similar in size. A total of
demonstrate inhibition of neutrophil Chemotaxis and cell 160 dentin chips were produced in this manner. The pulpal
lysis after treatment with Chlorhexidine. Helgeland et al.13 face of each dentin chip was flat and a reference mark was
showed that Chlorhexidine was toxic to cultures of human placed in the center with a #8 round bur in order to orient
epithelial cells and caused hemolysis of human erythro- this surface against the floor of the culture flasks.
cytes. Concentrations of Chlorhexidine well below those
used in clinical dentistry have been reported to cause cell Surface Area Analysis
injury, cell death, and inhibition of protein synthesis in The surface area available for fibroblast attachment on each
human fibroblast cultures and HELA cell cultures.14 of these pieces was determined by first covering the avail-
The adverse effects that Chlorhexidine has been reported able surface area (i.e., the surface area of the dentin chip
to have on fibroblasts may have significant impact on re- minus the area of the pulpal face of the dentin chip) with
generation therapy. The re-establishment of lost connective aluminum foil. Each piece of aluminum foil was weighed
tissue attachment to the root surface after periodontal ther- on a Cahn Electro-Balance and the area determined by ex-
apy is a desirable goal. The ability of periodontal ligament trapolation from a standard curve.
fibroblasts to repopulate root surfaces may be a critical The standard curve was prepared by weighing 10 alu-
event in the regeneration of connective tissue attachment minum foil squares in each of several known areas. There
on diseased root surfaces. This forms the basis for guided was a linear relationship between the weight of the alumi-
tissue regeneration techniques used in recent years.15 No num squares and their area. The teeth were stored in an
studies have been reported that evaluate the effect of Chlor- Ultra Low Freezer. Prior to each experiment the dentin
hexidine on fibroblast attachment to the surface of the root. chips were sterilized in a autoclave.
Determination of any effects that Chlorhexidine may have
on fibroblast attachment will provide valuable information Chlorhexidine Solution
for use of Chlorhexidine after periodontal surgery. The pur- A commercial preparation of 0.12% Chlorhexidine was used
pose of this study is to measure fibroblast attachment to in all experiments. The base contains water, 11.6% alcohol,
tooth surfaces treated with Chlorhexidine in vitro. glycerin, PEG-40 solution diisosterate, flavor, sodium, sac-
The null hypothesis is that Chlorhexidine has no effect charin, and FD + C Blue #1. An identical solution exclud-
on the attachment of human gingival fibroblasts to root ing Chlorhexidine was used in all control groups.
surfaces in vitro.
Experiment #1
MATERIALS AND METHODS In the first experiment the effect of exposing the dentin
chips to Chlorhexidine for 30 seconds, 90 seconds, or 3
Cell Cultures minutes intervals on fibroblast attachment was assessed.
Human gingival fibroblasts were obtained from stock cul- There were 3 experimental groups and 1 control group, each
tures at Medical College of Georgia School of Dentistry. containing 10 dentin chips randomly selected from the pool
These cells were maintained in plastic culture flasks fol- of 160 dentin chips. In the first experimental group the
lowing standard techniques. The cells were radioactively dentin chips were exposed to Chlorhexidine for 30 seconds,
labeled in culture to a standard specific activity using tri- in the second experimental group 90 seconds, and in the
tiated thymidine. Culture media consisted of RPMI 1640 third experimental group 3 minutes. The dentin chips in the
supplemented with 5% heat inactivated fetal calf serum, control group were exposed to the control solution for 3
0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 0.10 mg/ minutes. Following this incubation, all dentin chips were
ml neomycin, and 2.5 mg/ml amphotericin B. Cells were quickly rinsed in distilled water and fibroblasts were added
harvested using trypsin and used between the 5th and 10th to the culture vials for 1 hour.
cell passage.
Experiment #2
Collection of Teeth and Preparation of Root The second experiment was designed to determine if dentin
Fragments chips exposed to Chlorhexidine for 3 minutes had an effect
Fifty teeth were collected from the oral surgery clinic at the on fibroblast attachment with time. In this experiment there
Eisenhower Army Medical Center from patients undergoing were 4 experimental groups each paired with a control group.
routine extraction of impacted third molars. Only those teeth There were 10 dentin chips in each group. The 4 times of
that had been verified as having no communication with incubation of the fibroblasts were 2, 4, 6, and 8 hours. The
the oral cavity before extraction, and had not been sectioned dentin chips in all experimental groups were exposed to the
 J Periodontol
436 INHIBITION OF FIBROBLAST ATTACHMENT July 1991

Chlorhexidine solution for an interval of 3 minutes after

which they were rinsed in distilled water. The dentin chips
Effect of Chlorhexidine
in the control groups underwent identical treatment with the on Cell Attachment
control solution. The dentin chips in each group were then Thousands of Cells Attached
incubated with the fibroblast solutions for the various time
intervals.
6.8

Fibroblast Attachment
6.6

II.,
In both experiments, fibroblast attachment was measured
by exposing the dentin chips to a solution of fibroblasts (1 6.4
10 5 cells/ml) and allowed to incubate for the various
time intervals at 37°C. Unattached and loosely attached
6.2
cells were resuspended and removed by gently swirling in
a Gyrotory Shaker model G-2. After adding 10 ml of liquid
scintillation fluid, attached cells were sonicated for 15 sec- Control 30 Sec 90 Sec 180 Sec
onds using a Sonimer Cell Disruptor equipped with a mi- Time of Incubation
crotip and operated at an output control of 7. Radiolabeled Figure 1. The effect of Chlorhexidine on fibroblast attachment to dental
cells were then counted using a Beckman Scintillation Counter surfaces was assessed at different times of incubation. The dentinal chips
were exposed to Chlorhexidine for 30, 90, or 180 seconds before 1 hour
Model 8100. The number of cells per unit area were thus incubation with fibroblasts. Attachment is expressed as thousands of fi-
determined for each root fragment. broblasts attached. All 3 treatment times were significantly different from
control, but not significantly different from each other indicating that the
Statistical Analysis effect of Chlorhexidine was maximal after 30 second preineubation.
The data were analyzed by Repeated Measures Analysis
of Variance (RM-ANOVA) including group, treatment
and time main effects and group-by-treatment, group-by- Effect of CHX on Attachment
time, time-by-treatment, and group-by-time-by-treatment
interactions.
Cells/Sq mm
Salin« Control
in thousands
RESULTS Chlorhexidin«
12^
Effect of Time of Exposure to Chlorhexidine on
Fibroblast Attachment
In the first experiment, the dentin chips were exposed to 10J
Chlorhexidine for 30 seconds, 90 seconds, or 3 minutes
before being incubated with the fibroblasts for 1 hour.
There was a reduction in the number of fibroblast attach-
ment/mm squared of root surface in all 3 experimental
groups relative to the control group. (Fig. 1). Control
values were 6956 ± 617, whereas 30 second treatment
values were 6618 ± 829; 90 second 6695 ± 1014 and
3 minute 6456 ± 488. The difference between the ex- 4
Tim« (hour*)
perimental and the control groups is statistically signifi- Dentin chips were incubated with Chlorhexidine for 3 minutes,
cant at < 0.001. Chlorhexidine treatment of dentin Figure 2.
washed in PBS and incubated with fibroblasts for 2, 4, 6, and 8 hours.
chips resulted in a statistically significant reduction in PBS incubation served as control. Chlorhexidine exposure significantly
fibroblast attachment with a 1 hour incubation time, but inhibited fibroblast attachment at all time points.
it did not matter if the dentin chips were exposed to Chlor-
hexidine for 30 seconds, 90 seconds, or 3 minutes.
± 195 versus Chlorhexidine treated (CHX) at 6456 ± 154;
Effect of Incubation Time With Chlorhexidine-Treated 2 hours. C 7656 ± 281, CHX 7000 ± 345; 4 hours.
= =

Roots on Fibroblast Attachment C =

8276 ± 224, CHX 7403 ± 335; 6 hours C
= =

In the second experiment dentin chips which had been ex- 10459 ± 395, CHX 8654 ± 203 and 8 hours. C
= =

posed to Chlorhexidine for 3 minutes were incubated with 9809 ± 470, CHX 8207 ± 220. The within groups
=

the fibroblasts for 2, 4, 6, or 8 hours. The Chlorhexidine differences were statistically significant; i.e., time of in-
treatment resulted in a statistically (P < 0.001) significant cubation with fibroblasts had an effect on attachment since
reduction in fibroblast attachment relative to the controls at more fibroblasts attached with time. But the group-by-treat-
all time periods (Fig. 2). At 1 hour, control (C) was 6956 ment-by-time interaction was not significant. Therefore,
Volume 62
Number 7 ALLEYN, O'NEAL, STRONG, SCHEIDT, VAN DYKE, McPHERSON 437

Chlorhexidine resulted in a statistically significant reduction ulation tissue formation. This evidence is in contrast to the
in fibroblast attachment relative to the controls irrespective generally favorable results obtained in clinical studies.1-8 It
of time of incubation with fibroblasts. may be that the antimicrobial benefit of Chlorhexidine out-
weighs the cytotoxic side effects in wound healing post-
DISCUSSION periodontal surgery. On the other hand, clinical studies may
This study demonstrates that Chlorhexidine impairs fibro- not reflect the true picture and course of events at the his-
blast attachment to root surfaces in vitro. There was essen- tological level.
tially no difference whether or not the specimens were An important question that should be addressed is how
exposed to the Chlorhexidine solution for 30, 90, or 180 much Chlorhexidine penetrates into the wound site during
seconds. This suggests that the available binding sites in healing and actually contacts the fibroblasts in the connec-
the inorganic component of dentin (hydroxyapatite) or the tive tissue. One would think that this would depend on the
organic matrix (collagen) may have become saturated with mode of application of the drug, i.e., mouthrinsing vs.
Chlorhexidine within 30 seconds. The effect of Chlorhexi- direct irrigation with a syringe or an ultrasonic sealer. Sev-
dine was consistent in all experimental groups and persisted eral studies have explored this problem using disclosing
whether the incubation time with the fibroblasts was 1, 2, agents. Pitcher et al.19 found that mouthrinsing failed to
4, 6, or 8 hours. The implication of this finding is 2-fold. achieve any significant penetration of pockets but that direct
The first is that the effect of Chlorhexidine on fibroblast irrigation was partially effective. Hardy et al.20 demon-
attachment is for all intents and purposes manifest within strated that direct irrigation was effective in reaching the
the first hour of incubation with the cells. The second is apical plaque border in pockets. Eakle et al.21 showed the
that the effect of the drug is maintained over the time frame use of an oral irrigation designed for home use was able to
of this experiment. This is in keeping with the substantivity deliver solutions to approximately half the depth of the
of Chlorhexidine. Studies by Bonesvoll et al.16 have shown pockets on the average. All of these studies deal with depth
that 30% of a 10 ml 1 minute rinse of 0.2% Chlorhexidine of penetration into pockets as opposed to the depth of pen-
is bound in the oral cavity and is subsequently released over etration into the "wounded sulcus" immediately post-sur-
the next 8 to 12 hours. Indeed, weak concentrations were gically so it is not possible to extrapolate the results of these
found in saliva up to 24 hours. studies directly to this problem.
These results are in agreement with those of Goldschmidt If sufficient Chlorhexidine were able to come into contact
et al.14 in that concentrations as low as 0.004% Chlorhex- with the periodontal ligament cells during the initial stages
idine resulted in impaired cellular function and/or cell death. of healing one could rationalize that the drug may adversely
Exposure of fibroblasts to solutions of 0.12% Chlorhexidine affect regeneration of the attachment apparatus. This of
for as little as 30 seconds produced the same results. There course would be particularly true for guided tissue regen-
are some important differences between our study and theirs. eration techniques. It may be prudent to avoid forceful ir-
In the study by Goldschmidt et al.,14 the Chlorhexidine was rigation with Chlorhexidine using oral irrigators or irrigating
essentially incorporated into the growth medium of the fi- syringes until wound healing is well advanced.
broblasts at different concentrations and for varying time In conclusion, this study demonstrates that Chlorhexidine
periods. It could be argued that the effective concentrations adversely affects the attachment of human gingival fibro-
of Chlorhexidine that the fibroblasts were exposed to were blasts to root surfaces in vitro.
likely higher than what would be expected in vivo. In vivo
the fibroblasts are isolated by the epithelium from the oral Acknowledgements
environment and the flow of saliva and crevicular fluid Supported by the Clinical Investigations Division of Dwight
would tend to reduce the local concentrations of the drug. David Eisenhower Medical Center and USPHS Grants
In related studies, Nieders and Weiss17'18 demonstrated that DE06436 and DE07908.
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