312

Topical Application of Doxycycline on Periodontally Involved Root Surfaces in Vitro: Comparative Analysis of Substantivity on Cementum and Dentin Korkud Demirel, * Paul N.

Baer,f and Thomas F. McNamara*

In this study, substantivity of topically applied doxycycline hydrochloride on root surfaces obtained from patients with periodontal disease was studied in vitro. Cementum and dentin specimens were impregnated with aqueous solutions of doxycycline for 3 minutes, and incubated in serum for 10 minutes, 7 days, and 14 days. Substantivity was determined by agar diffusion inhibition assay. Comparative analyses of cementum versus dentin groups were conducted with 2-way analysis of variance. Results indicated that there were no statistically significant differences between the binding potential of doxycycline to cementum and dentin specimens. Antibacterial effect of specimens treated with the concentration of 100 mg/ml persisted for 14 days, indicating the presence of doxycyline in biologically active form. Our findings demonstrate the long lasting substantivity of doxycycline hydrochloride on periodontally diseased root surfaces and supports the concept of using root surfaces as a substrate for the deposition and slow release for local tetracycline delivery. J Periodontol 1991;62:312-316.

Key Words: Doxycycline/therapeutic

use;

tetracycline/therapeutic use; periodontal

dis-

eases/drug therapy.

One of the crucial problems subsequent to successful periodontal therapy is the recurrence of periodontopathic bacteria in previously treated sites. The root surfaces, particularly those which were in the periodontal pocket prior to treatment, probably play an important role in this recolonization. Bacteriological1 and histological2-3 studies have demonstrated that there is subsurface bacterial contamination of cementum and dentin along the periodontally involved root surfaces. In fact microorganisms have been found even within the cementum and/or dentin of those root surfaces which were aggressively scaled and root planed.3 In order to overcome this, attempts were made by various investigators to eradicate these bacterial reservoirs by topically applying antibacterial agents to the root surfaces.4"10 Tetracycline was the drug of choice because of its effectiveness against suspected causative microflora,11 its antienzymatic properties,12,13 and

'Department of Periodontics, School of Dental Medicine, S.U.N.Y, at Stony Brook, Stony Brook, NY and Department of Periodontology, University of Istanbul, Capa Istanbul, Turkey Department of Periodontics, School of Dental Medicine, S.U.N.Y, at Stony Brook, Stony Brook, NY. Department of Oral Biology and Pathology. -

of substantivity when applied topically to surfaces in vitro.5,14,15 A survey of the literature revealed that the existing in vitro studies on substantivity of tetracyclines on root surfaces were limited to dentin specimens obtained from teeth which had a clinically healthy periodontal environment. Consequently, the present investigation was designed to compare the substantivity of doxycycline hydrochloride on cementum and dentin specimens obtained from periodontally diseased teeth. its

high degree

root

MATERIALS AND METHODS of Specimens Cementum and dentin specimens were obtained from patients undergoing extraction of their permanent teeth due to advanced periodontal disease. The following clinical criteria were taken into consideration on sample selection: 1. Probing depths exceeding 5 mm at all 6 probing locations around each tooth; 2. Radiographic evidence of 50% or more alveolar bone

Preparation

loss;

3. No

history of antibiotic intake for at least 6 months;

Volume 62 Number 5

DEMIREL, BAER, McNAMARA

4. No history of any form of periodontal treatment during the preceeding 3 months; and 5. No root surface caries or restorations. Immediately after extraction the teeth were rinsed with sterile saline and stored in saline at 4°C until they were processed. One mm thick horizontal serial sections of the root portions that were exposed to the pocket flora were prepared by high speed water cooled diamond saw.§ Cementum specimens were prepared by planing the root surfaces under a dissection microscope with 13/14 Columbia curets to remove calculus and softened cementum from the root surfaces. Some specimens were excluded when it was visually determined that the cementum was not present. To prepare dentin specimens, the cementum was removed by water-cooled diamond burs to a depth of at least 0.25 mm, a depth which exceeds the average thickness of human cementum at the midportion of the root. After removing the cementum, dentin surfaces were also planed under a dissection microscope with 13/14 Columbia curets. Colored nail enamel was applied to the apical and coronal cut surfaces. The slabs were then sterilized by exposure to ethylene oxide gas, and stored in sterile saline at 4°C until used. Randomly selected cementum'6 and dentin specimens were observed under SEM to confirm the presence and continuity of cementum layer from the specimens addressed as dentin group. Doxycycline solutions of 100 mg/ml, 50 mg/ml, 10 mg/ ml, and 1 mg/ml were prepared in distilled water from doxycycline-HCLJ A 100 mg/ml concentration of doxycycline produced a solution of pH 2. This pH was maintained for the other 3 solutions by adding appropriate amounts of 12 HCL. Doxycycline solutions were then filter sterilized by disposible filters1' of 0.45 µ pore size. Sterile saline was selected as the control immersion solution. Cementum and dentin specimens were treated for 3 minutes with aqueous solutions of doxycycline. Following exposure to doxycycline at different concentrations, each of the test specimens was vortexed in 10 ml of sterile saline for 30 seconds to remove nonadsorbed doxycycline. Likewise control specimens were immersed in sterile saline for minutes and vortexed for 30 seconds. Control and test specimens were transferred to individual vials at 37°C for 10 minutes (Day 0), 7 or 14 days, all specimens were placed on a 1.5% Noble agar* surface layer in 100 mm petri dishes, and then overlayed with 8 ml of seeded agar. All experiments were run in triplicate. Bacterial Growth Inhibition Assay Actinomyces viscosas, Actinobacillus actinomycetemcomitants and Porphyromonas gingivalis were selected for study

sBronwill, Rochester, NY. "Sigma Chemical Co., St. Louis,

MO.

Sciences, Rochester, NY. 'Difco, Detroit, MI.

313

because of their association with periodontal disease. Stock cultures of A. viscosus (T14V), were transferred to Brain Heart Infusion (BHI) broth* which was supplemented with yeast extract (5 mg/ml), hemin solution (1%), and vitamin Kj (0.02%), Overnight growth of a broth culture under aerobic conditions at 37°C was adjusted to a 50% transmission at 700 nm producing a concentration of 2.5 x 105 bacteria/ml. A 10-fold dilution of this microbial suspension was used to seed BHI agar supplemented as above. This agar preparation was used to overlay the specimens. The results of agar diffusion inhibition assay were evaluated by measuring zones of inhibition following aerobic incubation at 37°C for 48 hours. Stock cultures oí A. actinomycetemcomitans (Y-4) were transferred to BHI broth which was supplemented with yeast extract (5 mg/ml), hemin solution (1%), vitamin (0.02%), and sodium bicarbonate (0.4%), and the organisms were incubated at 35°C for 48 hours in 5% C02 and 95% air environment. The culture was adjusted to 50% transmission at 700 nm which gave a bacterial population of 4 10s bacterial/ml. A 10-fold dilution of this bacterial suspension was used to seed supplemented BHI agar. Specimens were overlayed with this agar preparation and incubated at 35°C for 48 hours in 5% C02 and 95% air environment. Zones of inhibition of bacterial growth were then measured. P. gingivalis (381) stock cultures were transferred to BHI broth supplemented.17 Bacterial suspension was incubated at 37°C in an anaerobic chamber** for 48 hours. Bacterial growth was then adjusted to 50% transmission at 700 nm which gave a bacterial population of 5 106 bacteria/ml. A 10-fold dilution of this bacterial suspension was used to inoculate the supplemented BHI agar. Specimens were overlayed by this agar preparation and incubated anaerobically at 37°C for 48 hours. Following the incubation period, zones of inhibition were evaluated. The sensitivity of the test organisms to doxyclycline hydrochloride was conducted under similar conditions by placing 100 µ of 2-fold serial dilutions of doxycycline hydrochloride in standard size penicyclinders. Sensitivity was determined by measuring the zones of inhibition resulting after incubation for 48 hours. Evaluation and Statistical Analysis of the Results Peripheral edge of the specimens and inhibition zones were traced by a computer probe on an illuminated screen to determine the surface area. Then the surface area of the slabs was subtracted from the area of inhibition zones giving the true zone of inhibition for each slab. These zones were then divided by the surface area of the specimens which were exposed to doxycycline treatment in order to calculate the square area inhibited per square unit area of dentin or cementum. These data are expressed in units since they do not match with metric system.

TGelman

"Forma Scientific Model

L1020, Marietta, OH.

J Periodontol May 1991

EFFECT OF DOXYCYCLINE ON ROOT SURFACES IN VITRO

314



DENTIN



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DENTIN CEMENTUM

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LL

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i

io

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50

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RESULTS Observation of randomly selected specimens under SEM confirmed the presence and continuity of cementum in the group addressed as cementum, and total exposure of dentin in the dentin group. The pH of BHI agar supplemented with various additives were measured immediately before seeding with bacteria and found to be between the range of 6.8 to 7.2 Overall, throughout the study, control groups did not show zones of inhibition in the assay but demonstrated a slight reduction in the number of colony forming units (CFU) at the periphery of the specimens, which were not measurable, and were ignored throughout the evaluation of results. Figure 1 shows the results of agar diffusion-inhibition assay for A. viscosus. At day 0, inhibition zones created by impregnation of the specimens with solutions of 1 mg/ ml and 10 mg/ml concentrations were significantly (P 0.01) different for cementum versus dentin groups. Concentrations of 50 mg/ml also demonstrated a significant (P 0.05) difference between the cementum and dentin groups but the highest (100 mg/ml) concentration failed to elicit a significant difference. At day 7 there were no zones of inhibition for either group at the 1 mg/ml level. Doxycycline hydrochloride solutions at 10 mg/ml and 50 mg/ml did not show a significant difference between the zones of inhibition for the dentin and cementum groups, but at a concentration of 100 mg/ml the difference was significant at 0.1. only Zones of inhibition created by different concentrations =

=

=

Inc. Los

Angeles,

10

50

50

100

CA.

mg/ml

100

100

DAY 7

DAYO

Statistical evaluation of these results was conducted by 2-way analysis of variance (ANOVA) by BMDP7D tt program

Analysis Software,

1

DAY 14

DAY 7

Figure 1. The means and standard deviations of inhibition zones produced by cementum and dentin groups on agar plates seeded with A. viscosus. Zones of inhibition were calculated in unit square per each unit square of cementum or dentin specimen exposed to treatment with doxycycline hydrochloride.

1tBMDP 7 D Statistical

S-® DAY 14

2. The means and standard deviations of zones of inhibition produced by test groups with A. actinomycetemcomitans. Inhibition zones were calculated in V2IV2 of test specimen exposed to doxycycline treatment. Concentrations of impregnation solutions which did not demonstrate any zone of inhibition are not shown in the graph.

Figure

• DENTIN O CEMENTUM 20

O co

15

O

10

LU

0 1

io

so

DAYO

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50 loo

DAY 7

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50 loo

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DAY 14

Figure 3. The means and standard deviations of inhibition zones produced by test groups on plates seeded with P. gingivalis. Since the concentration of 100 mg/ml demonstrated full clearance at day Ofor both cementum and dentin groups, these results are not shown on the graph.

and time periods on agar plates seeded with A. actinomycetemcomitans are shown in Figure 2. Concentrations of 1 mg/ml and 10 mg/ml failed to create inhibition zones at days 7 and 14, but the 50 mg/ml group failed to show inhibition zones only at day 14. Unlike the experiments run with A. viscosus, there were no significant differences in the zones of inhibition between the cementum and dentin groups, although the dentin group showed slightly larger zones. The inhibition zones at day 0 increased with increasing concentrations of doxycycline, however, the differences were not statistically significant with the single exception of lmg/ml concentration of doxycycline. Similar results were observed on agar plates seeded with P. gingivalis (Fig. 3). The cementum and dentin groups revealed no significant differences, but as with the assays performed with other two microorganisms, the dentin group demonstrated greater zones of inhibition as compared to that

Volume 62 Number 5

DEMIREL, BAER, McNAMARA

315

for topical application in clinical trials. Furthermore, these results would also indicate that since the differences between cementum and dentin are negligible, and there is no rationale for total removal of cementum if it is being done solely to enhance substantivity. In the present in vitro study we have tried to simulate the clinical situation by subjecting the root specimens to daily changes of serum. It is recognized that in vitro drug testing may not always predict an in vivo outcome. Results of the sensitivity testing of the test organisms to doxyeycline hydrochloride are in agreement with previous use

. P. O

gingivalis

tz

m

a

A. viscosus

A.

O

actinomycetem-

comitans

ÚJ

o

io"

io

io

mg/ml

DOXYCYCLINE HCL CONCENTRATION

Figure

studied.

Sensitivity to doxyeycline hydrochloride of the microorganisms Diameters of inhibition zones are measured in cm.

4.

of the cementum group. Concentration of 100 mg/ml induced full clearance on agar plates at day 0, though these results were not considered in the statistical analysis. Zones of inhibition of the two higher concentrations were significantly (P 0.01) greater than the two lower concentrations when cementum and dentin groups were evaluated on time basis at day 7 and 14. Results of the sensitivity of the test organisms to doxyeycline hydrochloride revealed that A. actinomycetemcomitans was the most resistant, A. viscosus was moderately sensitive, and P. gingivalis was the most sensitive microorganism under conditions of this experiment (Fig. 4). =

DISCUSSION Roots obtained from areas adjacent to periodontal pockets have been shown to be contaminated with viable bacteria by microbiologie and microscopic methods.13 While in the past some investigators have suggested that endotoxins on the cementum might also play an important role in the etiology of periodontal disease more recent studies have tended to question those results and to rule out their importance.18 Clinically, the finding of viable bacteria on root surfaces certainly suggests that to obtain the best therapeutic results it would be advantageous to combine antibiotics with mechanical debridement. One of the main objectives of this study was to compare the substantivity of doxyeycline hydrochloride on diseaseaffected cementum and dentin. The dentin groups demonstrated large zones of inhibition throughout the study although these differences were not always statistically

significant. The highest concentration of doxyeycline (100 mg/ml) displayed inhibition zones with all three microorganisms at all time intervals, whereas the concentration of 50 mg/ ml failed to produce zones of inhibition at day 14 with A. açtinomycetemcomitans, the most resistant microorganism studied. This would suggest that the concentration of 100 mg/ml might be the most reasonable one to

tetracycline literature.11

We have demonstrated for the first time that cementum and dentin specimens obtained from periodontally diseased areas in humans are capable of acting as a reservoir by adsorbing and slowly releasing antibacterial levels of doxyeycline into the adjacent environment for several days fol-

lowing topical application of this antibiotic. REFERENCES 1. Adriens PA, DeBoever JA,

Loesche WJ. Bacterial invasion in root periodontally diseased teeth in hu-

cementum and radicular dentin of

/ Periodontol 1988;59:222. Daly CG, Seymour GJ, Kieser JB, Corbet EF. Histological assessment of periodontally involved cementum. / Clin Periodontol mans.

2.

1982;9:226.

3. Adriens PA, Edwards CA, DeBoever JA, Loesche WJ. Ultrastructural observations on bacterial invasion in cementum and radicular dentin of periodontally diseased human teeth. J Periodontol 1988;59:493. 4. Goodson JM, Haffajee A, Socransky SS. Periodontal therapy by local delivery of tetracycline. J Clin Periodontol 1979;6:83. 5. Baker PJ, Evans RT, Coburn RA, Genco RJ. Tetracycline and its derivatives strongly bind to and are released from the tooth surface in active form. / Periodontol 1983;54:580. 6. Bjorvatn K, Skaug N, Selvig KA. Inhibition of bacterial growth by tetracycline-impreghated enamel- and dentin. Scand J Dent Res

1984;92:508. M, Uematsu A, Nishijima K,

7. Minabe

et al. Application of a local drug delivery system to periodontal therapy I. Development of collagen preparations with immobilized tetracycline. / Periodontol

1989;60:113.

8. Minabe M, Takeuchi K, Tamura T, Hori T, Umemoto T. Subgingival administration of tetracycline on a collagen film. / Periodontol

1989;60:552.

9. Minabe M, Takeuchi K, Tomomatsu E, Hori T, Umemoto T. Clinical effects of local application of collagen film-immobilized tetracycline. J Clin Periodontol 1989;16:291. 10. Larsen T. In vitro release of doxyeycline from bioabsorbable materials and acrylic strips. J Periodontol 1990;61:30. 11. Baker PJ, Slots J, Genco RJ, Evans RT. Minimal inhibitory concentrations of various antimicrobial agents for human oral anaerobic bacteria. Antimicrob Agents Chemother 1983;24:420. 12. Golub LM, Ramamurthy TF, McNamara TF, et al. Tetracyclines inhibit tissue collagenase activity. A new mechanism in the treatment of periodontal disease. J Periodont Res 1984;19:651. 13. Gomes BC, Golub LM, Ramamurthy NS. Tetracyclines inhibit parathyroid hormone-induced bone résorption in organ culture. Experientia 1984;40:1273. 14. Bjorvatn K, Skaug N, Selvig KA. Tetracycline-impregnated enamel and dentin: Duration of antimicrobial capacity. Scand J Dent Res

1985;93:192.

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J Periodontol May 1991

EFFECT OF DOXYCYCLINE ON ROOT SURFACES IN VITRO

tetracycline-im-

penetration into root cementum of periodontally healthy and diseased

16. Zander HA, Hurzeler B. Continuous cementum apposition. / Dent Res 1958;37:1035. 17. Holdeman LV, Cato EP, Moore WEC. Anaerobe Laboratory Manual, 4th ed. Blacksburg, VA: Virginia Polytechnic Institute and State University; 1977:144. 18. Nakib NM, Bissada NF, Simmelink JW, Goldstine SN. Endotoxin

Send reprint requests to: Dr. Paul N. Baer, Professor and Chairman, Department of Periodontics, School of Dental Medicine, S.U.N.Y, at Stony Brook, Stony Brook, NY 11794-8703. Accepted for publication December 3, 1990.

15.

Bjorvatn K, Skaug N. Intraoral bacterial growth pregnated dentin. Scand J Dent Res 1986;94:95.

on

human teeth. J Periodontol

1982;53:368.

Topical application of doxycycline on periodontally involved root surfaces in vitro: comparative analysis of substantivity on cementum and dentin.

In this study, substantivity of topically applied doxycycline hydrochloride on root surfaces obtained from patients with periodontal disease was studi...
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