.................... .........................................*......

Joseph Shapira, DMD, Harold D. Sgan-Cohen, DMD, MPH, Ayala Stabholz, DMD, Michael N. Sela, DMD, PhD, Danny Schurr, MD, Josef Goultschin, DMD

Clinical and microbiological effects of chlorhexidine and arginine sustained-release varnishes in the mentally retarded

Local applications of sustained- released varnishes of chlorhexidine and arginine were used in a controlled pilot study of 34 mentally retarded patients, ages 18-45, assigned to one of these groups: chlorhexidine (C), arginine (A), or placebo (P). A professional scaling followed by four weeks of professional brushing to reach a Plaque Index (Pll) and Gingival Index (GI) of 1.0 at baseline preceded eight weeks of daily varnish application to the buccal and labial surfaces of all teeth. Clinical parameters (PI1 and GI) and bacterial samples from selected teeth were collected at predetermined intervals. Four and eight weeks following the baseline, the PI1 was significantly different among the groups, with the lowest score in the chlorhexidine group. No significant differences among the three groups were noted for the GI. The chlorhexidine and arginine groups showed significant reductions (p < 0.05 and p < 0.01, respectively) in the number of S. mutans. The arginine group showed a nonsignificant increase in the number of S. sanguis. These results suggest that the topical antimicrobial agents may have some relevance to plaque control among patients with mental retardation.

reatment of inflammatory periodontal diseases is based upon evidence that the

primary etiologic factor is bacterial plaque.’ The conventional approach to periodontal prevention and therapy, therefore, centers on plaque control. Mechanical oral hygiene maintenance can be obtained by most ordinary patients but not always by mentally and physically handicapped individuals. Frequently, these individuals are dependent on others to perform daily oral hygiene. Recently, there has been considerable interest in the use of antibacterial agents as an alternative measure for the control of dental plaque among people with disabilities. Previous studiess6 have shown that topical applications of kanamycin to tooth surfaces in institutionalized Down Syndrome patients have significantly reduced the plaque mass on the tooth surfaces compared with a placebo treatment. Chlorhexidine (CHX) mouthrinse has been shown to be effective in reducing the numbers of oral bacteria, thus playing an important role in plaque prevention among ordinary patients7’(’ as well as among patients with mental retardation.”,’?

New methods have been explored for the delivery of chemotherapeutic agents directly onto the supra- or subgingival area. Friedman and Golomb” have developed a topical sustained-release delivery system of chlorhexidine for prevention of plaque development. This delivery system overcomes several of the drug’s disadvantages when used conventionally. It reduces the dosage

and the frequency of use of chlorhexidine needed, and also reduces its side-effects. Moreover, this method does not necessitate the cooperation of the patient as does mouthrinsing, because the drug is applied by the therapist or by the person who supervises the patient’s oral hygiene. The effectiveness of the system has been shown in cl@?jcal trials using partial dentures, removable orthodontic appliances,”’ I’ and by direct application to the surfaces of the teeth.IH I ”

There is ample evidence that the ability of plaque organisms such as S. sanguis to utilize arginine (an amino acid) as a source of energy plays an important and even dominant role in their survival both in D~UO’” and in vitro.2’ While S. niutans cannot metabolize arginine, S. s a n p i s and S. milleri do ferment this substrate. The arginine catabolism proceeds in these streptococci via the arginine deiminase pathway with twice the release of ammonia.” This process of generating ammonia results in the reduction of pathogenic plaque bacteria and counteracts organic acids created by carbohydrate metabolism.?’ Experiments24 ’’ showed that arginine, when used alone or with urea, reduced plaque acidity, and De long and van der Hoeven’” found that arginine also has an effect on the plaque flora in favor of Streptococcus sanguis and Streptococcus milleri. When these organisms have been incubated with arginine, a rise in pH has been evident. The current investigation was designed as a pilot study to determine the clinical and microbiological effects of sustained-

158 Special Care in Dentistry, Vol14 No 4 1994

release delivery systems (SRD) of chlorhexidine and arginine on dental plaque in vivo, applied daily for eight weeks to the buccal and labial surfaces of the teeth of 34 mentally retarded patients with gingivitis.

Materials and methods Thirty-four mentally retarded individuals, living in one institution for retarded patients in the Jerusalem area, participated in the study. They included 11 females and 23 males between the ages of 18 and 45. After approval of the study and written consent from each patient's legal representative, the participants were selected from 240 residents of this institution, based on the following criteria:

(a) Healthy, with no history of other chronic illness except the mental deficiency.

(b) Ability to sit still on a dental chair during treatment.

(c) No dental treatment during the preceding three months. Supragingival and/or subgingival scaling was performed for all participants one month before study.

(d) Dentition including at least 20 functional teeth which were considered adequate for application of varnish and subsequent evaluations.

antibiotics two weeks prior to the study.

(e) No administration of

Due to the above restraints, only 34 subjects participated. The study population was randomly assigned to one of three groups:

Group 1: Experimental group C-chlorhexidine (11 subjects). Group 2: Experimental group A-arginine (11 subjects). Group 3: Control group P- placebo (12 subjects).

Table I. PI1 scores for buccal and lingual surfaces of all teeth.

Group Baseline One Week Two Weeks Four Weeks Eight Weeks (T") (T,) (T2) (TJ (T")

x SD x SD x SD x SD x SD

1.81 + 0.46 C 1.06 f 0.35 1.85 k 0.32 1.77f 0.35 1.74 f 0.48

A 0.78 f 0.32 1.75k 0.48 1.89 f 0.46 1.98 k 0.34 2.15 f 0.36

P 1.0 f 0.36 2.0 f 0.34 1.94 f 0.35 2.24 f 0.28 2.4 2 0.23

K- W N.S. N.S. N.S. p < 0.01 p < 0.002

M-W CHX-P1 p < 0.01 CHX-PI p < 0.002

Arg-PI p < 0.04

Group C = chlorhexidine Group A = Arginine Group P = Placebo

K-W = Kruskal-Wallis M-W = Mann-Whitney x = Mean SD = Standard Deviation

Prior to commencement of the study, the Plaque Index of Silness and L0e27 and the Gingival Index of Loe and Silness" were recorded for each patient. All patients underwent thorough scaling by a registered dental hygienist, followed by a daily brushing done by two senior dental students for four weeks until reaching P1I and GI scores of 1.0. NO oral hygiene instructions were given at any later stage of the study. The attendant team was instructed not to interfere with oral hygiene habits of the participants.

Within each assigned group, each individual received a three-digit reference number. The same number appeared on the bottle of a liquid polymer varnish assigned to the patient.

Varnish preparation

The sustained-release delivery varnishes (SRD) of chlorhexidine and arginine were prepared by embedding the active materials in a matrix of ethyl cellulose polymer as described by Friedman and Golomb.13 By adjusting the matrix structure, a prolonged and controlled drug release is attained for different periods of time. The drug release profile is modulated by varying initial drug concentrations and the addition of different casting solvents

into the matrix polymer. The active materials in the liquid polymer we used in the present study, based on the above release rate data, were either the chlorhexidine diacetate 1.6% or 3% arginine. The placebo liquid polymer varnish contained only solvents and polymer.

Varnish application

The liquid polymer consisted of a viscous solution which, when applied to the teeth, instantaneously produced a thin film attached to the surfaces of the teeth. After application of the liquid polymer on the tooth surface, the obtained film slowly degraded overnight. Each patient had the buccal and labial aspects of his/her teeth painted by a soft brush applicator, once a day, one hour after his/her last meal, for eight weeks. Applicants of the varnish were unaware of the content of the polymer. Following painting, the teeth were left to dry for 30 seconds until a thin white film was observed on the tooth surface. The active substance (whether chlorhex- idine or arginine) was released from the film in a sustained form, whereas the actual polymer was degraded by saliva during the night.

Clinical protocol

Clinical parameters (P1I and GI) and bacterial sampling were done at

Special Care in Dentistry, Vol 14 No 4 1994 159

Table 2. PI1 scores for the buccal surfaces of the anterior teeth at baseline,

4 weeks, 8 weeks. and increments. ~ ~~

Group Baseline Four Weeks Increment* Eight Weeks Increment**

(TJ (TI) (TA x SD x SD x SD

C 0.84 f 0.4 1.56 * 0.89 0.72 1.89 * 0.93 1.05

A 0.52 k 0.5 2.02 k 0.68 1.50 2.32 & 0.73 1 .so P 0.84 * 0.5 2.39 * 0.45 1.55 2.51 * 0.32 1.67

K-W N.S. N.S. p < 0.003 N.S. N.S. M-W CHX-P1 p < 0.02

Group C = chlorhexidine Group A = arginine Group P = placebo

*Difference between 4 weeks and baseline.

x = Mean SD = Standard Deviation

** Difference between 8 weeks and baseline.

baseline (immediately following professional tooth cleaning) (to) and one (t,), two (tJ, four (tJ, and eight (tH) weeks thereafter. The clinical indices were recorded from all teeth present in each mouth, both buccal and lingual surfaces, whereas bacterial samples were removed from selected teeth (7,10,13,24,27,29, and 30) and pooled. All clinical examinations were performed by one examiner (J.G.) in the morning after breakfast, with patients in the same dental chair and assigned to treatment groups without the examiner's knowledge. In this study, no side-effects were reported in all three groups.

Bacterial sampling

Supragingival plaque from the mesio-buccal, buccal, and distobuccal surfaces of the abovementioned selected teeth was removed by a 7/8 Gracey curette and placed on preweighed aluminum foils. Each sample was transferred into a sterile vial. Reduced transport fluid (RTF), which maintains the viability of the anaerobes in the samples until plated anaerobically, was added. A final concentration of 1 mg plaque/mL fluid was reached. The vials were then stored in ice and transferred to the microbiology laboratory.

seconds and then serially diluted in RTF (1:50; 1: lOO; 1:lOOO). A bacterial

The samples were vortexed for 60

sample from each dilution was plated on one of four media by means of an automatic spiral plater.

The selective media used were as follows: ETSA (Enriched Tryptic Soy Agar) for the selective growth of total anaerobic bacteria; MS (Mitis Salivarius agar) for the selective growth of total streptococci; MSB (Mitis Salivarius Bacitracin) for the selective growth of Streptococcus mutans; and TYC (Tryptic Yeast Casitone) for the selective growth of Streptococcus sangu is.

Following a 72-hour incubation under anaerobic conditions (ETSA) in an anaerobic chamber and facultative conditions (MS, MSB, TYC) in a candle jar, the total number of colony-forming units (CFU) was counted, and the mean values from the three dilutions further calculated in order to obtain the total bacterial count in 1 mg plaque weight.

Statistical analysis

The units of analysis for all variables examined were whole-mouth means. The mean values for each patient were calculated for each of the clinical indices as well as for the bacterial counts.

The Kruskal-Wallis one-way ANOVA was used to examine whether there were significant differences among the three study groups. In order to evaluate the effect

of the drug within each group, the changes from baseline for each examination time point (1,2,4, and 8 weeks) and the changes between any two examination time points were calculated. The Mann-Whitney U-test was used to distinguish which changes within groups were significantly different between groups. A probability level of p < 0.05 was considered significant.

Results

(1) Effect of the SRD on PI1 scores (Table 1)

At baseline, there was no significant difference between the mean P1I scores among the three groups. When the combined score of all buccal and lingual surfaces of all teeth examined is followed through the experimental period (8 weeks), in all three groups (C = CHX; A = Arginine; P = Placebo), a rise in the P1I scores was observed from time 0 to week 1. From week 2 to week 8, a moderate increase took place in the scores of all groups. At week 4, the lowest score was in the C group (Table 1). This score, PI1 = 1.74 f 0.48, was highly significant when compared with that of the placebo group (2.24 i 0.28; I' < 0.01). Group A score, P1I = 1.98 +- 0.34, compared with that of the placebo, was also significantly lower (P < 0.04). At week 8, the score of the C group (1.81 * 0.46) was significantly lower than that of group P (2.4 * 0.23; P < 0.002). Group A was not different from group P at eight weeks.

(2) PI1 scores for the buccal surfaces of the anterior teeth only (Table 2)

All three groups showed a very gradual increase from baseline through the end of the experimental period. Here too, the lowest scores were in the C group. When the differences among the mean P1I values for each of the three groups were compared between baseline (To) and 4 weeks (T,) and 8 weeks (TJ, highly significant differences among the groups were noted only between T,l and 4 weeks, with a significant difference between the CHX and the

160 Special Care in Dentistry, Vol14 No 4 1994

placebo groups (I' < 0.02).

(3) PI1 scores for buccal surfaces of the lower anterior teeth (Table 3)

Significant differences for mean P1I values between baseline (TJ and 4 weeks (T,) and 8 weeks (TJ were noted for the lower anterior teeth only between CHX and placebo groups.

(4) Gingival Index (GI) scores for buccal and lingual surfaces of all teeth (Table 4)

From time 0 and throughout the study, there was a gradual rise of the Gingival Index in all groups. No significant differences of the mean values between the different time intervals were noted for the three groups.

(5) Microbiology (Table 5)

No statistically significant reduction in the number of S. viridans, S. mutans, or S. sanguis between weeks 1 and 5 of the placebo group could be observed.

The chlorhexidine group, on the other hand, showed a statistically significant reduction in the number of S. mutans (from 1.54 x lob to 7.96 x lo', p < 0.05). No change was observed in this group with regard to S. viridans and S . sanguis. On the other hand, the arginine group showed a rise in the number of S. sanguis, a reduction in the number of S. viridans, and a significant reduction in the number of S. mutans (from 3.26 x lo6 to 7.2 x lo', p < 0.01).

Discussion

The use of conventional oral hygiene methods among institutionalized mentally retarded individuals is often inadequate. The effect of this care is dependent on the motivation and capability of the institution

This study, therefore, explored an alternative method of maintaining adequate oral hygiene among an institutionalized population using sustained-release varnishes. We assume that the distribution of existing inadequate self oral hygiene measures was equal

Table 3. PI1 scores for the buccal surfaces of the lower anterior teeth at baseline, 4 weeks, 8 weeks, and increments.

Group Baseline Four Weeks Increment* Eight Weeks Increment**

(TJ (T'J (TJ x SD x SD x SD

C 0.97 f 0.5 1.45 f 0.94 0.48 1.79 * 0.92 0.82

A 0.54 ? 0.48 2.12 * 0.65 1.58 2.25 ? 0.74 1.70

P 0.93 k 0.69 2.31 ? 0.79 1.38 2.61 f 0.59 1 .68

K-W N.S. N.S p < 0.01 N.S. p < 0.05

MW CHX-PI D < 0.01 CHX-PI D < 0.04

*Difference between 4 weeks and baseline.

x = Mean SD = Standard Deviation

** Group C = chlorhexidine Group A = arginine Group P = placebo

Difference between 8 weeks and baseline.

among the three study groups, due to the random design. One week after commencement of the study (T,), a sharp rise in the P1I scores of all groups was noted, due to termination of supervised daily brushing by dental students. Four weeks following the baseline, the plaque indices were significantly lower among the chlorhexidine and arginine groups, as compared with the placebo group. After 8 weeks, only the chlorhexidine group demonstrated a significantly lower plaque level.

McKenzie,12 who evaluated the use of a 0.12% CHX mouthrinse among the institutionalized mentally handicapped, found a significant improvement in the P1I and GI among the CHX group after one month, but this returned to baseline after 12 months. Cutressl" evaluated the use of CHX-containing dentifrice gel by mentally disabled over a six- month period and found no clinical improvement in the Gingival Index. He concluded that periodontal severity and poor oral hygiene exceeded the therapeutic potential of CHX. Brayer,?& in a CHX rinsing study among mentally retarded, demonstrated a minimal reduction in GI scores compared with the P1I scores among the CHX group. He concluded that the plaque-preventing

effect of CHX is due to its interference with the bacterial absorption mechanism, thus reducing the number of bacteria locally on the tooth surface and not around the gingival sulcus area. Flotra" conducted a four-month trial comparing the effects of mouthrinses containing 0.1% or 0.2% CHX applied twice daily as a supplement to normal oral hygiene. After eight weeks, the CHX significantly reduced plaque and gingival scores by 66% and 31% respectively. Two similar studies," i4 performed on normal populations, reached the same results. Their conclusions are very similar: "CHX mouthrinse can provide an important adjunct to the prevention and control of gingivitis, when used with regular personal oral hygiene procedures and professional care."'4 In addition, the ability of CHX to control gingivitis is related to its ability to control total plaque quantity and periodontopathic microorganisms in the plaque."

direct bactericidal activity, the local application of arginine to supragingival plaque is an effective means of generating ammonia by a catabolic process occurring in some streptococcus strains found in dental plaque, especially S. sanguis.2225"' The pH of plaque, which is usually acidic,

In contrast to CHX, that has strong

Special Care in Dentistry, Vol 14 No 4 1994 161

Table 4. GI scores for buccal and lingual aspects of all teeth.

Group Baseline One Week Two Weeks Four Weeks Eight Weeks

no) (TI) (T2) (T4) (TR) x SD x SD x SD x SD x SD

C 1.14 & 0.17 1.26 + 0.19 1.33 * 0.26 1.35 * 0.22 1.44 k 0.33

A 1.18 * 0.16 1.30 f 0.21 1.41 * 0.26 1.57 + 0.24 1.47 * 0.22

P 1.09 * 0.19 1.25 k 0.16 1.28 f 0.20 1.44 ? 0.17 1.37 * 0.22

K- W N.S. N.S. N.S. N.S. N.S.

Group C = chlorhexidine Group A = arginine Group P = Placebo x = Mean SD = Standard deviation

Table 5. The effects of chlorhexidine and arginine on the numbers of Colony-Forming units (CFU) in plaque bacteria.

PlaceboChlorhexidine Arginine week 1 5

week 1 5

week 1 5

S. viridans 417,363 186,456 78,000 150,000 756,291 213,318

S. niufaris 1,345,454 451,908 1,547,500 796,363* 3,260,000 723,813**

S. sanauis 155,928 126,176 9,680 6,940 47,009 132,534

* = p < 0.05 ** = p < 0.01

is raised by the ammonia, and this in turn has long-term effects in reducing pathogenic bacteria, especially S. mutans, which is pH-sensitive.zS,'h

of a sustained-release delivery system of arginine on the buccal and labial aspects of subjects' teeth increased the proportions of S . sanguis in the dental plaque, and showed a highly statistically significant reduction in the number of pathogenic S. mutans. These results are in accordance with the hypothesis that bacterial nutrients (such as arginine) are important ecological factors in the dental plaque environment.2h The modification of plaque to improve its resistance

In the present study, application

against pH fall and increase of pathogenic bacteria is most desirable and encouraging without the use of an antibacterial agent over long periods of time.

The present study population was not necessarily optimal for a clinical study. A larger and more cooperative group would have been preferable, but the basic premise was that a potential target population for the daily use of chlorhexidine and arginine varnishes would be a mentally disabled population. Due to the size of the population, these results should be regarded only within the context of a pilot study.

The personnel operated to the best of their ability, but we are aware that

at the time of the varnish application the cooperation between the therapist and tired subjects (at night before sleep) was not always optimal. Under the conditions of this study, where plaque accumulation rate was decreased over time (plaque was not eliminated or reduced), it should not have been expected that GI would improve, as was the finding in the present study.

antimicrobial agents are not total substitutes for mechanical removal of plaque but may serve as important adjuncts to personal oral hygiene measures.

In conclusion, topical

The authors gratefully thank the staff at the Elwin Institute for Mentally Retarded Patients, in Jerusalem, for their cooperation with this study, with special thanks to Dr. M. Finnelt and the dental hygienist, Ms. Ofra Eliahu, without whose assistance this work could have not been performed. We also express our appreciation to Perio Products Co. Ltd., for providing the materials for this study and for financial support. This work was done in partial fulfillment of the requirements for the DMD degree by Drs. Osher Marilena and Tal Nurit, who were dental students at that time.

Dr. Shapira is Associate Clinical Professor, Department of Pediatric Dentistry; Dr. Sgan- Colien is Senior Lecturer, Department of Community Dentistry; Dr. Stabholz is Clinical Senior Lecturer, Department of Community Dentistry; Dr. Sela is Professor, Department of Oral Biology; Dr. Goultschin is Associate Clinical Professor, Department of Periodontology, all from the Faculty of Dental Medicine, Hebrew University-Hadassah Medical Center, P.O. Box 1172, Jerusalem 91010, Israel. Dr. Schurr is Medical Director, Elwin Institute, Jerusalem. Correspondence should be addressed to Dr. Shapira.

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