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-establishment 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 fibroblast 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 periodontal therapy due to its bactericidal effect. Chlorhexidine is a cationic molecule and belongs to the polybiguanide group of compounds. The bactericidal effect of the drug results from the cationic molecule altering the osmotic equilibrium of the microbes.1 The effectiveness of Chlorhexidine rinse as a plaque control agent is due largely to its substantivity. It adsorbs on oral surfaces and then is slowly released in active form.2 Numerous studies suggest the use of Chlorhexidine mouthrinse has a favorable effect on plaque control and gingival inflammation. Lindhe et al.3 showed •U.S. Army, Ft. Gordon, GA. tCurrently, Eastman Dental Center, Rochester, NY; previously, Emory University, Atlanta, GA. The opinions expressed in this article do not represent the views of the United States Department of Defense, the Department of Army, or the United States Dental Corps. Use of any commercial products in this project does not imply endorsement by the U.S. Government.
that in dogs treatment with Chlorhexidine resulted in resolution of gingivitis and significantly lowered plaque scores. Bogle et al.4 demonstrated greater bone, regeneration in bifurcation defects in dogs when topical Chlorhexidine was used post operatively. Langenbaek and Bay5 showed a reduction of plaque index and gingival exúdate when a Chlorhexidine mouthrinse was used after gingivectomy. A study by Bakaeen and Strahan6 suggests that there may be less postoperative pain when Chlorhexidine is used after periodontal surgery, although they were unable to show any significant differences in Plaque Index, Gingival Index, crevicular fluid, or depth of pockets. Newman and Addy7 were able to demonstrate a reduction in plaque scores and sulcus bleeding in patients who used Chlorhexidine postoperatively, but found no significant reduction of postoperative pain. A study by Asboe-Jorgensen et al.8 revealed a reduction in gingival exúdate and a decreased tendency to bleeding following postoperative use of Chlorhexidine.
Volume 62 Number 7
ALLEYN, O'NEAL, STRONG, SCHEIDT, VAN DYKE, McPHERSQN
In contrast to the above, several studies provide evidence demonstrating toxic effects of Chlorhexidine on human cells and granulation tissue. Paunio et al.9 and Bassetti and Tallenberger10 demonstrated that Chlorhexidine delays granulation tissue formation. Cytotoxicity has been demon-
strated in neutrophils.11,12 These studies were able to demonstrate inhibition of neutrophil Chemotaxis and cell lysis after treatment with Chlorhexidine. Helgeland et al.13 showed that Chlorhexidine was toxic to cultures of human epithelial cells and caused hemolysis of human erythrocytes. Concentrations of Chlorhexidine well below those used in clinical dentistry have been reported to cause cell injury, cell death, and inhibition of protein synthesis in human fibroblast cultures and HELA cell cultures.14 The adverse effects that Chlorhexidine has been reported to have on fibroblasts may have significant impact on regeneration therapy. The re-establishment of lost connective tissue attachment to the root surface after periodontal therapy is a desirable goal. The ability of periodontal ligament fibroblasts to repopulate root surfaces may be a critical event in the regeneration of connective tissue attachment on diseased root surfaces. This forms the basis for guided tissue regeneration techniques used in recent years.15 No studies have been reported that evaluate the effect of Chlorhexidine on fibroblast attachment to the surface of the root. Determination of any effects that Chlorhexidine may have on fibroblast attachment will provide valuable information for use of Chlorhexidine after periodontal surgery. The purpose of this study is to measure fibroblast attachment to tooth surfaces treated with Chlorhexidine in vitro. The null hypothesis is that Chlorhexidine has no effect on the attachment of human gingival fibroblasts to root surfaces in vitro.
435
during extraction, were used. After removing any debris on
the root surface with a sealer, the crowns were removed and the cementum removed with a diamond bur. Removal of cementum was confirmed visually using a stereomicroscope. The roots were then sectioned into dentin chips which were rectangular in shape and similar in size. A total of 160 dentin chips were produced in this manner. The pulpal face of each dentin chip was flat and a reference mark was placed in the center with a #8 round bur in order to orient this surface against the floor of the culture flasks. Surface Area Analysis The surface area available for fibroblast attachment on each of these pieces was determined by first covering the available surface area (i.e., the surface area of the dentin chip minus the area of the pulpal face of the dentin chip) with aluminum foil. Each piece of aluminum foil was weighed on a Cahn Electro-Balance and the area determined by extrapolation from a standard curve. The standard curve was prepared by weighing 10 aluminum foil squares in each of several known areas. There was a linear relationship between the weight of the aluminum squares and their area. The teeth were stored in an Ultra Low Freezer. Prior to each experiment the dentin chips were sterilized in a autoclave. Chlorhexidine Solution A commercial preparation of 0.12% Chlorhexidine was used in all experiments. The base contains water, 11.6% alcohol, glycerin, PEG-40 solution diisosterate, flavor, sodium, saccharin, and FD + C Blue #1. An identical solution excluding Chlorhexidine was used in all control groups.
Experiment #1
MATERIALS AND METHODS Cell Cultures Human gingival fibroblasts were obtained from stock cultures at Medical College of Georgia School of Dentistry. These cells were maintained in plastic culture flasks following standard techniques. The cells were radioactively labeled in culture to a standard specific activity using tritiated thymidine. Culture media consisted of RPMI 1640 supplemented with 5% heat inactivated fetal calf serum, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 0.10 mg/ ml neomycin, and 2.5 mg/ml amphotericin B. Cells were harvested using trypsin and used between the 5th and 10th cell passage.
Collection of Teeth and
Fragments
Preparation
of Root
Fifty teeth were collected from the oral surgery clinic at the Eisenhower Army Medical Center from patients undergoing routine extraction of impacted third molars. Only those teeth that had been verified as having no communication with the oral cavity before extraction, and had not been sectioned
In the first experiment the effect of exposing the dentin chips to Chlorhexidine for 30 seconds, 90 seconds, or 3 minutes intervals on fibroblast attachment was assessed. There were 3 experimental groups and 1 control group, each containing 10 dentin chips randomly selected from the pool of 160 dentin chips. In the first experimental group the dentin chips were exposed to Chlorhexidine for 30 seconds, in the second experimental group 90 seconds, and in the third experimental group 3 minutes. The dentin chips in the control group were exposed to the control solution for 3 minutes. Following this incubation, all dentin chips were quickly rinsed in distilled water and fibroblasts were added to the culture vials for 1 hour.
Experiment #2
The second experiment was designed to determine if dentin chips exposed to Chlorhexidine for 3 minutes had an effect on fibroblast attachment with time. In this experiment there were 4 experimental groups each paired with a control group. There were 10 dentin chips in each group. The 4 times of incubation of the fibroblasts were 2, 4, 6, and 8 hours. The dentin chips in all experimental groups were exposed to the
436
J Periodontol July 1991
INHIBITION OF FIBROBLAST ATTACHMENT
Chlorhexidine solution for an interval of 3 minutes after which they were rinsed in distilled water. The dentin chips in the control groups underwent identical treatment with the control solution. The dentin chips in each group were then incubated with the fibroblast solutions for the various time intervals. Fibroblast Attachment In both experiments, fibroblast attachment was measured by exposing the dentin chips to a solution of fibroblasts (1 10 5 cells/ml) and allowed to incubate for the various time intervals at 37°C. Unattached and loosely attached 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 seconds using a Sonimer Cell Disruptor equipped with a microtip and operated at an output control of 7. Radiolabeled cells were then counted using a Beckman Scintillation Counter Model 8100. The number of cells per unit area were thus determined for each root fragment.
Statistical Analysis The data were analyzed by Repeated Measures Analysis of Variance (RM-ANOVA) including group, treatment and time main effects and group-by-treatment, group-bytime, time-by-treatment, and group-by-time-by-treatment interactions. RESULTS Effect of Time of Exposure to Chlorhexidine on Fibroblast Attachment In the first experiment, the dentin chips were exposed to 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 attachment/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 experimental and the control groups is statistically signifi< 0.001. Chlorhexidine treatment of dentin cant at chips resulted in a statistically significant reduction in fibroblast attachment with a 1 hour incubation time, but it did not matter if the dentin chips were exposed to Chlorhexidine for 30 seconds, 90 seconds, or 3 minutes. Effect of Incubation Time With Chlorhexidine-Treated Roots on Fibroblast Attachment In the second experiment dentin chips which had been exposed to Chlorhexidine for 3 minutes were incubated with the fibroblasts for 2, 4, 6, or 8 hours. The Chlorhexidine treatment resulted in a statistically (P < 0.001) significant reduction in fibroblast attachment relative to the controls at all time periods (Fig. 2). At 1 hour, control (C) was 6956
Effect of Chlorhexidine on
Cell Attachment
Thousands of Cells Attached
6.8
6.6
6.4
6.2
Control
II., 90 Sec
30 Sec
180 Sec
Time of Incubation Figure 1. The effect of Chlorhexidine on fibroblast attachment to dental surfaces was assessed at different times of incubation. The dentinal chips were exposed to Chlorhexidine for 30, 90, or 180 seconds before 1 hour incubation with fibroblasts. Attachment is expressed as thousands of fibroblasts attached. All 3 treatment times were significantly different from control, but not significantly different from each other indicating that the effect of Chlorhexidine was maximal after 30 second preineubation.
Effect of CHX Cells/Sq
on
mm
Attachment Salin« Control
in thousands
Chlorhexidin«
12^
10J
4
Tim« (hour*)
Dentin chips were incubated with Chlorhexidine for 3 minutes, washed in PBS and incubated with fibroblasts for 2, 4, 6, and 8 hours. PBS incubation served as control. Chlorhexidine exposure significantly inhibited fibroblast attachment at all time points.
Figure 2.
± 195 versus Chlorhexidine treated (CHX) at 6456 ± 154; 2 hours. C 7656 ± 281, CHX 7000 ± 345; 4 hours. 7403 ± 335; 6 hours C C 8276 ± 224, CHX 8654 ± 203 and 8 hours. C 10459 ± 395, CHX 9809 ± 470, CHX 8207 ± 220. The within groups differences were statistically significant; i.e., time of incubation with fibroblasts had an effect on attachment since more fibroblasts attached with time. But the group-by-treatment-by-time interaction was not significant. Therefore, =
=
=
=
=
=
=
=
Volume 62 Number 7
ALLEYN, O'NEAL, STRONG, SCHEIDT, VAN DYKE, McPHERSON
Chlorhexidine resulted in a statistically significant reduction in fibroblast attachment relative to the controls irrespective of time of incubation with fibroblasts. DISCUSSION This study demonstrates that Chlorhexidine impairs fibroblast attachment to root surfaces in vitro. There was essentially no difference whether or not the specimens were exposed to the Chlorhexidine solution for 30, 90, or 180 seconds. This suggests that the available binding sites in the inorganic component of dentin (hydroxyapatite) or the organic matrix (collagen) may have become saturated with Chlorhexidine within 30 seconds. The effect of Chlorhexidine was consistent in all experimental groups and persisted whether the incubation time with the fibroblasts was 1, 2, 4, 6, or 8 hours. The implication of this finding is 2-fold. The first is that the effect of Chlorhexidine on fibroblast attachment is for all intents and purposes manifest within the first hour of incubation with the cells. The second is that the effect of the drug is maintained over the time frame of this experiment. This is in keeping with the substantivity of Chlorhexidine. Studies by Bonesvoll et al.16 have shown that 30% of a 10 ml 1 minute rinse of 0.2% Chlorhexidine is bound in the oral cavity and is subsequently released over the next 8 to 12 hours. Indeed, weak concentrations were found in saliva up to 24 hours. These results are in agreement with those of Goldschmidt et al.14 in that concentrations as low as 0.004% Chlorhexidine resulted in impaired cellular function and/or cell death. Exposure of fibroblasts to solutions of 0.12% Chlorhexidine for as little as 30 seconds produced the same results. There are some important differences between our study and theirs. In the study by Goldschmidt et al.,14 the Chlorhexidine was essentially incorporated into the growth medium of the fibroblasts at different concentrations and for varying time periods. It could be argued that the effective concentrations of Chlorhexidine that the fibroblasts were exposed to were likely higher than what would be expected in vivo. In vivo the fibroblasts are isolated by the epithelium from the oral environment and the flow of saliva and crevicular fluid would tend to reduce the local concentrations of the drug. In related studies, Nieders and Weiss17'18 demonstrated that Chlorhexidine inhibits Ehrlich ascites cell division and adhesion to enamel. In our study the dentin fragments were exposed to a 0.12% solution of Chlorhexidine rinsed in distilled water, and only then were the dentin chips incubated with the fibroblasts. In this way, we hoped to get an appreciation of the substantivity of Chlorhexidine. As a result the only Chlorhexidine that the fibroblasts were exposed to was that which was released from the dentin chips into the growth medium. No attempt was made to determine the concentrations of Chlorhexidine in the growth medium after the dentin chips were placed in solution. Several studies9-14 provide evidence that Chlorhexidine exhibits cytotoxic effects to human cells and delays gran-
437
ulation tissue formation. This evidence is in contrast to the generally favorable results obtained in clinical studies.1-8 It may be that the antimicrobial benefit of Chlorhexidine outweighs the cytotoxic side effects in wound healing postperiodontal surgery. On the other hand, clinical studies may not reflect the true picture and course of events at the histological level. An important question that should be addressed is how much Chlorhexidine penetrates into the wound site during healing and actually contacts the fibroblasts in the connective tissue. One would think that this would depend on the mode of application of the drug, i.e., mouthrinsing vs. direct irrigation with a syringe or an ultrasonic sealer. Several studies have explored this problem using disclosing agents. Pitcher et al.19 found that mouthrinsing failed to achieve any significant penetration of pockets but that direct irrigation was partially effective. Hardy et al.20 demonstrated that direct irrigation was effective in reaching the apical plaque border in pockets. Eakle et al.21 showed the use of an oral irrigation designed for home use was able to deliver solutions to approximately half the depth of the pockets on the average. All of these studies deal with depth of penetration into pockets as opposed to the depth of penetration into the "wounded sulcus" immediately post-surgically so it is not possible to extrapolate the results of these studies directly to this problem. If sufficient Chlorhexidine were able to come into contact with the periodontal ligament cells during the initial stages of healing one could rationalize that the drug may adversely affect regeneration of the attachment apparatus. This of course would be particularly true for guided tissue regeneration techniques. It may be prudent to avoid forceful irrigation with Chlorhexidine using oral irrigators or irrigating syringes until wound healing is well advanced. In conclusion, this study demonstrates that Chlorhexidine adversely affects the attachment of human gingival fibroblasts to root surfaces in vitro.
Acknowledgements
Supported by the Clinical Investigations Division of Dwight
David Eisenhower Medical Center and USPHS Grants DE06436 and DE07908. REFERENCES 1. Greenstein G, Berman C, Jaffin R. Chlorhexidine, an adjunct to peri-
therapy. J Periodontol 1986; 57:370. Lang N, Brecx M. Chlorhexidine digluconate an agent for chemical plaque control and prevention of gingival inflammation. / Periodont Res 1986; 21(suppl.):74. Lindhe J, Hamp S, Loe , Rindom-Schiott C. Influence of topical application of Chlorhexidine on chronic gingivitis and gingival wound healing in the dog. Scand J Dent Res 1970; 78:471. Bogle G, Rathbun E, Oliver R, Hornbuckle C, Egelburg J. Effect of post-operative use of Chlorhexidine on regeneration of bifurcation defects in dogs. J Periodont Res 1974; 9:127. Langenbaek J, Bay L. The effect of Chlorhexidine mouthrinse on healing after gingivectomy. Scand J Dent Res 1976; 84:224. Bakaeen G, Strahan J. Effects of a 1% Chlorhexidine gel during the odontal
2.
—
3.
4.
5. 6.
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7.
8.
9.
10.
11.
12. 13. 14.
15.
J Periodontol July 1991
INHIBITION OF FIBROBLAST ATTACHMENT
healing phase after inverse bevel mucogingival flap surgery. J Clin Periodontol 1980; 7:20. Newman P, Addy M. Comparison of hypertonic saline and Chlorhexidine mouthrinses after the inverse bevel flap procedure. J Periodontol 1982; 53:315. Asboe-Jorgensen V, Attstrom R, Lang N, Loe H. Effect of a Chlorhexidine dressing on the healing after periodontal surgery. / Periodontol 1974; 45:13. Paunio K, Knuttila M, Mielityinen H. The effect of Chlorhexidine gluconate on the formation of experimental granulation tissue. J Periodontol 1978; 49:92. Bassetti C, Tallenburger A. Influence of Chlorhexidine rinsing on the healing of oral mucosa and osseous lesions J Clin Periodontol 1980; 7:443. Gabler W, Bullock W, Creamer H. The influence of Chlorhexidine on Superoxide generation by induced human neutrophils. J Periodont Res 1987; 22:445. Watts T, Addison , Johnson . Effects of Chlorhexidine solution on neutrophil locomotion in vitro. J Dent 1989; 17:287. Helgeland , Heyden G, Rolla G. Effect of Chlorhexidine on animal cells in vitro. Scandi Dent Res 1971; 79:209. Goldschmidt , Cogen R, Taubman S. Cytopathologic effects of Chlorhexidine on human cells. J Periodontol 1977; 48:212. Gottlow J, Nyman S, Karring T, Lindhe J. New attachment formation
result of controlled tissue regeneration. / Clin Periodontol 1984; 11:494. Bonesvoll P, Lokken P, Rolla G, and Pavs P. Retention of Chlorhexidine in the human oral cavity after mouth rinses. Arch Oral Biol 1974; 19:209. Neiders ME, Weiss L. The effect of Chlorhexidine treatment on the electrokinetic characteristics of enamel and cell adhesion to human enamel in vitro. Arch Oral Biol 1972; 17:949. Neiders ME, Weiss L. The effects of Chlorhexidine on cell attachment in vitro. Arch Oral Biol 1972; 17:961. Pitcher G, Newman H, Strahan J. Access to subgingival plaque by disclosing agents using mouthrinse and direct irrigation. J Clin Periodontol 1980; 7:300. Hardy J, Newman H, Strahan J. Direct irrigation and subgingival plaque. J Clin Periodontol 1982; 9:57. Eakle W, Ford C, Boyd R. Depth of penetration in periodontal pockets with oral irrigation. / Clin Periodontol 1986; 13:39. Taylor A, Campbell M. Reattachment of gingival epithelium to the tooth. / Periodontol 1972; 43:281.
as a
16.
17.
18. 19.
20. 21. 22.
Send reprint requests to: Director, Periodontal Residency Program, U.S. Army Dental Activity, Ft. Gordon, GA 30905. Accepted for publication January 18, 1991.
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-establishment 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 fibroblast 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 periodontal therapy due to its bactericidal effect. Chlorhexidine is a cationic molecule and belongs to the polybiguanide group of compounds. The bactericidal effect of the drug results from the cationic molecule altering the osmotic equilibrium of the microbes.1 The effectiveness of Chlorhexidine rinse as a plaque control agent is due largely to its substantivity. It adsorbs on oral surfaces and then is slowly released in active form.2 Numerous studies suggest the use of Chlorhexidine mouthrinse has a favorable effect on plaque control and gingival inflammation. Lindhe et al.3 showed •U.S. Army, Ft. Gordon, GA. tCurrently, Eastman Dental Center, Rochester, NY; previously, Emory University, Atlanta, GA. The opinions expressed in this article do not represent the views of the United States Department of Defense, the Department of Army, or the United States Dental Corps. Use of any commercial products in this project does not imply endorsement by the U.S. Government.
that in dogs treatment with Chlorhexidine resulted in resolution of gingivitis and significantly lowered plaque scores. Bogle et al.4 demonstrated greater bone, regeneration in bifurcation defects in dogs when topical Chlorhexidine was used post operatively. Langenbaek and Bay5 showed a reduction of plaque index and gingival exúdate when a Chlorhexidine mouthrinse was used after gingivectomy. A study by Bakaeen and Strahan6 suggests that there may be less postoperative pain when Chlorhexidine is used after periodontal surgery, although they were unable to show any significant differences in Plaque Index, Gingival Index, crevicular fluid, or depth of pockets. Newman and Addy7 were able to demonstrate a reduction in plaque scores and sulcus bleeding in patients who used Chlorhexidine postoperatively, but found no significant reduction of postoperative pain. A study by Asboe-Jorgensen et al.8 revealed a reduction in gingival exúdate and a decreased tendency to bleeding following postoperative use of Chlorhexidine.
Volume 62 Number 7
ALLEYN, O'NEAL, STRONG, SCHEIDT, VAN DYKE, McPHERSQN
In contrast to the above, several studies provide evidence demonstrating toxic effects of Chlorhexidine on human cells and granulation tissue. Paunio et al.9 and Bassetti and Tallenberger10 demonstrated that Chlorhexidine delays granulation tissue formation. Cytotoxicity has been demon-
strated in neutrophils.11,12 These studies were able to demonstrate inhibition of neutrophil Chemotaxis and cell lysis after treatment with Chlorhexidine. Helgeland et al.13 showed that Chlorhexidine was toxic to cultures of human epithelial cells and caused hemolysis of human erythrocytes. Concentrations of Chlorhexidine well below those used in clinical dentistry have been reported to cause cell injury, cell death, and inhibition of protein synthesis in human fibroblast cultures and HELA cell cultures.14 The adverse effects that Chlorhexidine has been reported to have on fibroblasts may have significant impact on regeneration therapy. The re-establishment of lost connective tissue attachment to the root surface after periodontal therapy is a desirable goal. The ability of periodontal ligament fibroblasts to repopulate root surfaces may be a critical event in the regeneration of connective tissue attachment on diseased root surfaces. This forms the basis for guided tissue regeneration techniques used in recent years.15 No studies have been reported that evaluate the effect of Chlorhexidine on fibroblast attachment to the surface of the root. Determination of any effects that Chlorhexidine may have on fibroblast attachment will provide valuable information for use of Chlorhexidine after periodontal surgery. The purpose of this study is to measure fibroblast attachment to tooth surfaces treated with Chlorhexidine in vitro. The null hypothesis is that Chlorhexidine has no effect on the attachment of human gingival fibroblasts to root surfaces in vitro.
435
during extraction, were used. After removing any debris on
the root surface with a sealer, the crowns were removed and the cementum removed with a diamond bur. Removal of cementum was confirmed visually using a stereomicroscope. The roots were then sectioned into dentin chips which were rectangular in shape and similar in size. A total of 160 dentin chips were produced in this manner. The pulpal face of each dentin chip was flat and a reference mark was placed in the center with a #8 round bur in order to orient this surface against the floor of the culture flasks. Surface Area Analysis The surface area available for fibroblast attachment on each of these pieces was determined by first covering the available surface area (i.e., the surface area of the dentin chip minus the area of the pulpal face of the dentin chip) with aluminum foil. Each piece of aluminum foil was weighed on a Cahn Electro-Balance and the area determined by extrapolation from a standard curve. The standard curve was prepared by weighing 10 aluminum foil squares in each of several known areas. There was a linear relationship between the weight of the aluminum squares and their area. The teeth were stored in an Ultra Low Freezer. Prior to each experiment the dentin chips were sterilized in a autoclave. Chlorhexidine Solution A commercial preparation of 0.12% Chlorhexidine was used in all experiments. The base contains water, 11.6% alcohol, glycerin, PEG-40 solution diisosterate, flavor, sodium, saccharin, and FD + C Blue #1. An identical solution excluding Chlorhexidine was used in all control groups.
Experiment #1
MATERIALS AND METHODS Cell Cultures Human gingival fibroblasts were obtained from stock cultures at Medical College of Georgia School of Dentistry. These cells were maintained in plastic culture flasks following standard techniques. The cells were radioactively labeled in culture to a standard specific activity using tritiated thymidine. Culture media consisted of RPMI 1640 supplemented with 5% heat inactivated fetal calf serum, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 0.10 mg/ ml neomycin, and 2.5 mg/ml amphotericin B. Cells were harvested using trypsin and used between the 5th and 10th cell passage.
Collection of Teeth and
Fragments
Preparation
of Root
Fifty teeth were collected from the oral surgery clinic at the Eisenhower Army Medical Center from patients undergoing routine extraction of impacted third molars. Only those teeth that had been verified as having no communication with the oral cavity before extraction, and had not been sectioned
In the first experiment the effect of exposing the dentin chips to Chlorhexidine for 30 seconds, 90 seconds, or 3 minutes intervals on fibroblast attachment was assessed. There were 3 experimental groups and 1 control group, each containing 10 dentin chips randomly selected from the pool of 160 dentin chips. In the first experimental group the dentin chips were exposed to Chlorhexidine for 30 seconds, in the second experimental group 90 seconds, and in the third experimental group 3 minutes. The dentin chips in the control group were exposed to the control solution for 3 minutes. Following this incubation, all dentin chips were quickly rinsed in distilled water and fibroblasts were added to the culture vials for 1 hour.
Experiment #2
The second experiment was designed to determine if dentin chips exposed to Chlorhexidine for 3 minutes had an effect on fibroblast attachment with time. In this experiment there were 4 experimental groups each paired with a control group. There were 10 dentin chips in each group. The 4 times of incubation of the fibroblasts were 2, 4, 6, and 8 hours. The dentin chips in all experimental groups were exposed to the
436
J Periodontol July 1991
INHIBITION OF FIBROBLAST ATTACHMENT
Chlorhexidine solution for an interval of 3 minutes after which they were rinsed in distilled water. The dentin chips in the control groups underwent identical treatment with the control solution. The dentin chips in each group were then incubated with the fibroblast solutions for the various time intervals. Fibroblast Attachment In both experiments, fibroblast attachment was measured by exposing the dentin chips to a solution of fibroblasts (1 10 5 cells/ml) and allowed to incubate for the various time intervals at 37°C. Unattached and loosely attached 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 seconds using a Sonimer Cell Disruptor equipped with a microtip and operated at an output control of 7. Radiolabeled cells were then counted using a Beckman Scintillation Counter Model 8100. The number of cells per unit area were thus determined for each root fragment.
Statistical Analysis The data were analyzed by Repeated Measures Analysis of Variance (RM-ANOVA) including group, treatment and time main effects and group-by-treatment, group-bytime, time-by-treatment, and group-by-time-by-treatment interactions. RESULTS Effect of Time of Exposure to Chlorhexidine on Fibroblast Attachment In the first experiment, the dentin chips were exposed to 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 attachment/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 experimental and the control groups is statistically signifi< 0.001. Chlorhexidine treatment of dentin cant at chips resulted in a statistically significant reduction in fibroblast attachment with a 1 hour incubation time, but it did not matter if the dentin chips were exposed to Chlorhexidine for 30 seconds, 90 seconds, or 3 minutes. Effect of Incubation Time With Chlorhexidine-Treated Roots on Fibroblast Attachment In the second experiment dentin chips which had been exposed to Chlorhexidine for 3 minutes were incubated with the fibroblasts for 2, 4, 6, or 8 hours. The Chlorhexidine treatment resulted in a statistically (P < 0.001) significant reduction in fibroblast attachment relative to the controls at all time periods (Fig. 2). At 1 hour, control (C) was 6956
Effect of Chlorhexidine on
Cell Attachment
Thousands of Cells Attached
6.8
6.6
6.4
6.2
Control
II., 90 Sec
30 Sec
180 Sec
Time of Incubation Figure 1. The effect of Chlorhexidine on fibroblast attachment to dental surfaces was assessed at different times of incubation. The dentinal chips were exposed to Chlorhexidine for 30, 90, or 180 seconds before 1 hour incubation with fibroblasts. Attachment is expressed as thousands of fibroblasts attached. All 3 treatment times were significantly different from control, but not significantly different from each other indicating that the effect of Chlorhexidine was maximal after 30 second preineubation.
Effect of CHX Cells/Sq
on
mm
Attachment Salin« Control
in thousands
Chlorhexidin«
12^
10J
4
Tim« (hour*)
Dentin chips were incubated with Chlorhexidine for 3 minutes, washed in PBS and incubated with fibroblasts for 2, 4, 6, and 8 hours. PBS incubation served as control. Chlorhexidine exposure significantly inhibited fibroblast attachment at all time points.
Figure 2.
± 195 versus Chlorhexidine treated (CHX) at 6456 ± 154; 2 hours. C 7656 ± 281, CHX 7000 ± 345; 4 hours. 7403 ± 335; 6 hours C C 8276 ± 224, CHX 8654 ± 203 and 8 hours. C 10459 ± 395, CHX 9809 ± 470, CHX 8207 ± 220. The within groups differences were statistically significant; i.e., time of incubation with fibroblasts had an effect on attachment since more fibroblasts attached with time. But the group-by-treatment-by-time interaction was not significant. Therefore, =
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Volume 62 Number 7
ALLEYN, O'NEAL, STRONG, SCHEIDT, VAN DYKE, McPHERSON
Chlorhexidine resulted in a statistically significant reduction in fibroblast attachment relative to the controls irrespective of time of incubation with fibroblasts. DISCUSSION This study demonstrates that Chlorhexidine impairs fibroblast attachment to root surfaces in vitro. There was essentially no difference whether or not the specimens were exposed to the Chlorhexidine solution for 30, 90, or 180 seconds. This suggests that the available binding sites in the inorganic component of dentin (hydroxyapatite) or the organic matrix (collagen) may have become saturated with Chlorhexidine within 30 seconds. The effect of Chlorhexidine was consistent in all experimental groups and persisted whether the incubation time with the fibroblasts was 1, 2, 4, 6, or 8 hours. The implication of this finding is 2-fold. The first is that the effect of Chlorhexidine on fibroblast attachment is for all intents and purposes manifest within the first hour of incubation with the cells. The second is that the effect of the drug is maintained over the time frame of this experiment. This is in keeping with the substantivity of Chlorhexidine. Studies by Bonesvoll et al.16 have shown that 30% of a 10 ml 1 minute rinse of 0.2% Chlorhexidine is bound in the oral cavity and is subsequently released over the next 8 to 12 hours. Indeed, weak concentrations were found in saliva up to 24 hours. These results are in agreement with those of Goldschmidt et al.14 in that concentrations as low as 0.004% Chlorhexidine resulted in impaired cellular function and/or cell death. Exposure of fibroblasts to solutions of 0.12% Chlorhexidine for as little as 30 seconds produced the same results. There are some important differences between our study and theirs. In the study by Goldschmidt et al.,14 the Chlorhexidine was essentially incorporated into the growth medium of the fibroblasts at different concentrations and for varying time periods. It could be argued that the effective concentrations of Chlorhexidine that the fibroblasts were exposed to were likely higher than what would be expected in vivo. In vivo the fibroblasts are isolated by the epithelium from the oral environment and the flow of saliva and crevicular fluid would tend to reduce the local concentrations of the drug. In related studies, Nieders and Weiss17'18 demonstrated that Chlorhexidine inhibits Ehrlich ascites cell division and adhesion to enamel. In our study the dentin fragments were exposed to a 0.12% solution of Chlorhexidine rinsed in distilled water, and only then were the dentin chips incubated with the fibroblasts. In this way, we hoped to get an appreciation of the substantivity of Chlorhexidine. As a result the only Chlorhexidine that the fibroblasts were exposed to was that which was released from the dentin chips into the growth medium. No attempt was made to determine the concentrations of Chlorhexidine in the growth medium after the dentin chips were placed in solution. Several studies9-14 provide evidence that Chlorhexidine exhibits cytotoxic effects to human cells and delays gran-
437
ulation tissue formation. This evidence is in contrast to the generally favorable results obtained in clinical studies.1-8 It may be that the antimicrobial benefit of Chlorhexidine outweighs the cytotoxic side effects in wound healing postperiodontal surgery. On the other hand, clinical studies may not reflect the true picture and course of events at the histological level. An important question that should be addressed is how much Chlorhexidine penetrates into the wound site during healing and actually contacts the fibroblasts in the connective tissue. One would think that this would depend on the mode of application of the drug, i.e., mouthrinsing vs. direct irrigation with a syringe or an ultrasonic sealer. Several studies have explored this problem using disclosing agents. Pitcher et al.19 found that mouthrinsing failed to achieve any significant penetration of pockets but that direct irrigation was partially effective. Hardy et al.20 demonstrated that direct irrigation was effective in reaching the apical plaque border in pockets. Eakle et al.21 showed the use of an oral irrigation designed for home use was able to deliver solutions to approximately half the depth of the pockets on the average. All of these studies deal with depth of penetration into pockets as opposed to the depth of penetration into the "wounded sulcus" immediately post-surgically so it is not possible to extrapolate the results of these studies directly to this problem. If sufficient Chlorhexidine were able to come into contact with the periodontal ligament cells during the initial stages of healing one could rationalize that the drug may adversely affect regeneration of the attachment apparatus. This of course would be particularly true for guided tissue regeneration techniques. It may be prudent to avoid forceful irrigation with Chlorhexidine using oral irrigators or irrigating syringes until wound healing is well advanced. In conclusion, this study demonstrates that Chlorhexidine adversely affects the attachment of human gingival fibroblasts to root surfaces in vitro.
Acknowledgements
Supported by the Clinical Investigations Division of Dwight
David Eisenhower Medical Center and USPHS Grants DE06436 and DE07908. REFERENCES 1. Greenstein G, Berman C, Jaffin R. Chlorhexidine, an adjunct to peri-
therapy. J Periodontol 1986; 57:370. Lang N, Brecx M. Chlorhexidine digluconate an agent for chemical plaque control and prevention of gingival inflammation. / Periodont Res 1986; 21(suppl.):74. Lindhe J, Hamp S, Loe , Rindom-Schiott C. Influence of topical application of Chlorhexidine on chronic gingivitis and gingival wound healing in the dog. Scand J Dent Res 1970; 78:471. Bogle G, Rathbun E, Oliver R, Hornbuckle C, Egelburg J. Effect of post-operative use of Chlorhexidine on regeneration of bifurcation defects in dogs. J Periodont Res 1974; 9:127. Langenbaek J, Bay L. The effect of Chlorhexidine mouthrinse on healing after gingivectomy. Scand J Dent Res 1976; 84:224. Bakaeen G, Strahan J. Effects of a 1% Chlorhexidine gel during the odontal
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healing phase after inverse bevel mucogingival flap surgery. J Clin Periodontol 1980; 7:20. Newman P, Addy M. Comparison of hypertonic saline and Chlorhexidine mouthrinses after the inverse bevel flap procedure. J Periodontol 1982; 53:315. Asboe-Jorgensen V, Attstrom R, Lang N, Loe H. Effect of a Chlorhexidine dressing on the healing after periodontal surgery. / Periodontol 1974; 45:13. Paunio K, Knuttila M, Mielityinen H. The effect of Chlorhexidine gluconate on the formation of experimental granulation tissue. J Periodontol 1978; 49:92. Bassetti C, Tallenburger A. Influence of Chlorhexidine rinsing on the healing of oral mucosa and osseous lesions J Clin Periodontol 1980; 7:443. Gabler W, Bullock W, Creamer H. The influence of Chlorhexidine on Superoxide generation by induced human neutrophils. J Periodont Res 1987; 22:445. Watts T, Addison , Johnson . Effects of Chlorhexidine solution on neutrophil locomotion in vitro. J Dent 1989; 17:287. Helgeland , Heyden G, Rolla G. Effect of Chlorhexidine on animal cells in vitro. Scandi Dent Res 1971; 79:209. Goldschmidt , Cogen R, Taubman S. Cytopathologic effects of Chlorhexidine on human cells. J Periodontol 1977; 48:212. Gottlow J, Nyman S, Karring T, Lindhe J. New attachment formation
result of controlled tissue regeneration. / Clin Periodontol 1984; 11:494. Bonesvoll P, Lokken P, Rolla G, and Pavs P. Retention of Chlorhexidine in the human oral cavity after mouth rinses. Arch Oral Biol 1974; 19:209. Neiders ME, Weiss L. The effect of Chlorhexidine treatment on the electrokinetic characteristics of enamel and cell adhesion to human enamel in vitro. Arch Oral Biol 1972; 17:949. Neiders ME, Weiss L. The effects of Chlorhexidine on cell attachment in vitro. Arch Oral Biol 1972; 17:961. Pitcher G, Newman H, Strahan J. Access to subgingival plaque by disclosing agents using mouthrinse and direct irrigation. J Clin Periodontol 1980; 7:300. Hardy J, Newman H, Strahan J. Direct irrigation and subgingival plaque. J Clin Periodontol 1982; 9:57. Eakle W, Ford C, Boyd R. Depth of penetration in periodontal pockets with oral irrigation. / Clin Periodontol 1986; 13:39. Taylor A, Campbell M. Reattachment of gingival epithelium to the tooth. / Periodontol 1972; 43:281.
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Send reprint requests to: Director, Periodontal Residency Program, U.S. Army Dental Activity, Ft. Gordon, GA 30905. Accepted for publication January 18, 1991.
