Diffusion of Tetracycline-Impregnated Gutta-Percha Points

ABSTRACT

The purpose of this study was to use an enzyme immunoassay to determine whether diffusion of tetracycline occurs through root dentin of extracted human teeth. Twenty-one roots were mechanically prepared and then divided into three groups: experimental (G1; n=15; tetracycline-impregnated gutta-percha (TGP) with MCS sealer); negative control (G2; n=3; plain gutta percha with MCS sealer) and MCS control (G4; n=3; TGP without sealer). All roots were coronally sealed and then immersed in saline. Three additional samples were used as the positive control (G3; n=3; one free TGP cone n saline). Results showed significantly more tetracycline diffusion between Group 3 and all other groups. There was no significant difference among Groups 1, 2 and 4. Maximum diffusion was seen at 24 hours, however, the rate of diffusion dropped off significantly. Teeth obturated with TGP and MCS sealer did not show any diffusion of tetracycline above an average of 0.006 g/ml within the time frames tested.

Pathways of disease transmission between the periodontium and the root canal system may occur through patent lateral and accessory canals, dentinal tubules or apical formamina.1 Simon et al. (1972) proposed a classification of endodontic-periodontic lesions and suggested that treatment of these teeth require both endodontic and periodontic therapy.2 As endodontic treatment is very effective when performed properly, the prognosis will usually depend on the amount of bony support lost from the accompanying periodontitis and the ability of periodontal treatment to resolve the residual defect.3 Periodontal therapy may include thorough root debridement, systemic or local delivery of antibiotics and antiseptics, root resection, regenerative therapy or forced eruption.

Systemic and locally delivered tetracycline has been widely used in the treatment of various types of periodontal diseases that are refractory.4 It has a broad spectrum of activity against many gram-positive and gram-negative bacteria. Systemic delivery of tetracycline tends to concentrate in bone and teeth due to its ability to bind to mineralized dentinal matrices and its slow release from dentin makes its antimicrobial effect substantive. Repeated doses of oral tetracylcine have been found to achieve gingival crevicular fluid levels that are two to four times that of blood levels, thus creating a reservoir above the minimal inhibitory concentration for most pathogenic bacteria found in the oral cavity.4-6

Moreover, tetracycline, unlike most alkaline antimicrobials, is stable in acidic environments and remains effective in inflamed periradicular tissue.7 Tetracycline has also been found to inhibit collagenase activity and thus bone resorbtion.

A widely used locally delivered periodontal fiber impregnated with tetracycline is Actisite (Alza Corp., Palo Alto, CA). These fibers are inserted subgingivally into periodontal pockets to further increase concentrations in the gingival crevicular fluid and can sustain concentrations in excess of 1300 g/ml for 10 days,5 a concentration which is well beyond the minimal inhibitory concentration (MIC) range of 0.016-2.000 g/ml needed to inhibit growth of 90 percent of periodontal and endodontic pathogens in vitro.8 Studies show that combined therapy of scaling and root planing plus tetracycline fibers results in significantly less sites of disease progression after a year of monitoring when compared to scaling and root planing alone.9 Moreover, local delivery of tetracycline minimizes the potential for developing drug resistance.10

From an endodontic standpoint, iodoform-containing gutta percha, MGP (Lone Star Technologies, Westport, CT) and more recently, tetracycline-impregnated gutta percha (TGP), (Medidenta International Inc., Woodside, NY) have been patented by Dr. Howard Martin in order to prevent bacterial contamination after endodontic treatment7 (Fig. 1). Furthermore, Dr. Martin recommends using his TGP in combination with his patented iodoform-containing zinc oxide eugenol sealer, MCS, (Lone star technologies, Westport, CT) when obturating teeth with long standing infections.11

Studies have shown that many techniques of sealer application only manage to cover 25% to 75% of canal walls.12 Moreover, sealers have been shown to resorb with prolonged exposure to tissue fluid.13 It is thus likely that portals of exit remain patent through which periodontal and periapical tissues may come into direct contact with TGP. This source of moisture would theoretically activate TGP and cause the release of tetracycline. The tetracycline would then diffuse through and binds to lateral canals or dentinal tubules to act as a reservoir of substantive tetracycline.7

Given tetracycline’s ability to diffuse through and bind to dentin,7 TGP may have the potential to be used as a new local drug delivery system in clinical situations requiring both endodontic and periodontal therapy.

The purpose of this study was to use an enzyme immunoassay to determine whether diffusion of tetracycline occurs through root dentin of extracted human teeth obturated with TGP and MCS sealer.

MATERIALS AND METHODS

Phase 1: Specimen preparation and saline sample collection

Twenty-one recently extracted human teeth were selected for the study and stored in tap water. The teeth ranged in type from centrals to second molars. The crowns were removed, all carious lesions were eliminated and multi-rooted teeth were sectioned into single roots. No attempt was made to preserve the periodontal ligament. All individual roots were modified at the coronal aspect to create 13mm root specimens (Figs. 2A&B). All specimens were autoclaved for 30 minutes at 250F at 15 PSI prior to cleaning and shaping. One operator prepared each specimen aseptically using rotary instrumentation with 0.6 taper Profile series 29 (Dentsply Tulsa Dental, USA). The apical foramen was enlarged to 0.35mm. Five ml of 6.0% sodium hypochlorite was used throughout the cleaning and shaping procedure. Seventeen per cent EDTA was allowed to soak for one minute in all prepared canals to remove the smear layer. All canals were dried with paper points and randomly divided into experimental and control groups. All specimens were obturated using cold lateral condensation.

The experimental group (G1) consisted of 15 specimens that were obturated with tetracycline-impregnated gutta percha (TGP) and MCS sealer. Group two (G2) was the negative control and comprised of three specimens that were obturated using non-medicated gutta percha and MCS sealer. Group 3 (G3) was the positive control and consisted of three freestanding TGP cones. In group four (G4), three specimens were obturated using TGP without sealer. The coronal aspect of all root specimens were conditioned with 37% phosphoric acid gel (Gel Etchant, Kerr, Orange, CA) for 15 seconds, thoroughly rinced for five seconds and lightly dried by gentle air blowing. OptiBond SOLO (Kerr, Orange, CA) bonding system was applied according to the manufacturer’s instructions and light cured for 20 seconds. Charisma restorative material (Heraeus-Kulzer, Hanau, Germany), shade A3, was then applied in stages to the bonded surface and light cured for 40 seconds after each increment.

Following obturation, G1, G2 and G4 samples were immediately stored in 100% humidity for 24 hr and then transferred into individually labelled, sealed and sterilized 15ml plastic test tubes filled with 10ml of sterile 0.9% saline. At this time, G3 freestanding TGP cones were placed into their individual test tubes filled with 10ml of sterile 0.9% saline. All test tubes were wrapped in aluminium foil to minimize exposure to light. The tubes were then placed in an incubator at 37C for specific time intervals: 24 hr, 48 hr, 96 hr, 1 week and 4 weeks.

At the end of each time interval both test and control specimens were removed from their tubes containing the saline solution and the tubes were rewrapped in aluminum and stored in the freezer. All specime
ns were then placed into new individually labelled sealed and sterilized 15ml plastic test tubes filled with 10ml of fresh, sterile 0.9% saline. Aluminum was used again to wrap each tube. All new tubes were once again placed in the incubator until the next time interval under study at which time this process was repeated..

Phase 2: Ridascreen tetracyclin competitive ELISA (R-Biopharm Inc, South Marshall, MI)

A pilot study was first completed in order to determine what dilution of each test and control saline sample was required to produce a concentration in the range between 0.15 and 1.35g/kg or parts per billion (ppb). This concentration range corresponds to the linear portion of the calibration curve shown in Graph 1. A series of standard tetracycline concentrates of 0, 0.05, 0.15, 0.45, 1.35 and 4.05 ppb in a buffer solution (provided in the Ridacreen Tetracyclin kit) are used to produce the calibration curve. The standard concentrates are run along with all the samples.14

Each Ridascreen kit contains 96 microtiter wells with one sample per well. Each sample is tested in duplicate. Four Ridascreen kits were used in total and the wells were arranged in the microtiter holders provided. Fifty l of standard, experimental and control samples were added to each well. Fifty l of anti-tetracycline antibody solution was then added to each well and the solutions were gently mixed and left to sit for one hour at room temperature. After one hour, the solutions were poured out of each well and all residual liquid was removed by tapping the microwell holders upside down on a paper towel. The wells were then washed with PBS buffer with Tween. One hundred l of enzyme conjugate was then added and gently mixed. This solution was allowed to sit for 15 minutes at room temperature. After 15 minutes, the solutions were again poured out, and all residual liquid was removed by tapping the microwell holders upside down on a paper towel. The wells were washed again with PBS buffer with tween and 50l of chromogen was added to each well. The wells were gently mixed and left to sit for 15 minutes in the dark. After 15 minutes, 100l of 1N sulphuric acid stop reagent was added and gently mixed. The absorbance was measured at 450nm using a microtiter plate spectrophotometer and is inversely proportional to the concentration of tetracycline in the standards or samples. An air blank was used for comparison. The absorbance was read within 60 minutes of adding the stop reagent. The concentration of tetracycline in g/kg in each sample was then read from the calibration curve using the mean values of absorbance obtained. The actual concentration of tetracycline in each sample was further multiplied by a corresponding dilution factor and converted to g/ml.14

STATISTICAL ANALYSIS

Data was collected and stored in a spreadsheet program and coded for statistical analysis. Data calculation was preformed using SYSTAT v11 for Windows (SYSTAT Software, Inc, Point Richmond, CA). Both descriptive statistics and two-factor ANOVA were carried out.

RESULTS

Tables 1 through 4 show diffusion concentrations for tetracycline in all samples for each group and at each time interval. At an alpha level of 0.05, the results of a two-factor ANOVA indicated that for every time interval studied, only the positive control, G3, had significant tetracycline diffusion, (28.486g/ml) relative to the experimental (G1: 0.006 g/ml) and other control groups (G4: 0.075g/ml, G2: 0.000g/ml). The difference in diffusion concentrations of tetracycline for G1, G2 and G4 were not significant (Table 5, Table 6 & Graph 2). When the concentration of tetracycline was assessed through time, it was found that maximum tetracycline diffusion occurred at 24 hours (17.190g/ml). Diffusion dropped to a minimum at 96 hours (2.676g/ml) and rose sharply by 4 weeks (6.860g/ml) (Table 7, Graph 3).

DISCUSSION

Local delivery of tetracycline and its derivatives have long been used as adjunctive therapy in the treatment of various types of periodontitis. Periodontitis associatied with endodontic lesions presents a unique situation where the root canal system can be used to locally deliver an effective antimicrobial to the periodontium via various patent pathways such as dentinal tubules or lateral canals. Both placement of an intra-appointment medicament or the final obturation are opportunities to provide local delivery of a drug.

The present study was the first to use an enzyme immunoassay, Ridacreen Tetracyclin (R-Biopharm Inc.), to investigate whether tetracycline diffusion occurs through root dentin of extracted human teeth when obturated with TGP, MCS sealer and coronally sealed. In the past, authors such as Wikesjo et al15 and Ciarlone et al16 used radioactive tetracycline and liquid scintillation counting to quantify the amount of tetracycline released from dentin.

Enzyme immunoassays are frequently used in dental research to quantify various molecules such as pro-inflammatory cytokines17 or endogenous beta-endorphin.18

Ridacreen Tetracyclin is a commercially available enzyme immunoassay. It is commonly used to detect tetracycline residues in meat, milk, honey and water samples as this antibiotic, among others, is used as a supplement in animal feed to accelerate the growth of food-producing livestock.19 According to the manufacturer, this enzyme immunoassay can detect tetracycline concentrations as low as 0.5 x 10-5 g/ml.14

Tetracycline is also known to exhibit photodegradation.20 To minimize its molecular breakdown, the diffused tetracycline in all saline samples was shielded from possible light sources by individually wrapping each sample tube with aluminium foil.

Tetracycline is also known to break down when dissolved in various liquids.10 Thus the concentration of tetracycline detected in the experimental and control saline solutions may have been inactive biologic moieties from parent tetraciclyine molecules. In an attempt to prevent or at least delay this occurrence, the experts at R-Biopharm Inc. recommended that all samples be immediately frozen. Apart from the actual diffusion of complete or partial tetracycline molecules in solution, the antimicrobial efficacy of the diffused tetracyline cannot be acertained based on the results of this study. Further research would be required to confirm the antibacterial effect of the diffused tetracycline.

TGP and MGP arm gutta percha with a broad spectrum of antibacterial efficacy. This is pertinent in situations where coronal leakage after obturation, leading to bacterial contamination and reinfection. Alternatively, TGP and MGP, could offer resistance against residual bacteria left in the root canal system that may otherwise produce persistent disease.17 Moisture contamination is purported to activate tetracycline and iodine from TGP and MGP respectively.21,7 Melker et al (2006) found TGP to be superior to both MGP and regular gutta percha against bacteria commonly found in failing root canal treatments, including E.faecalis.22 Moreover, Dr. Martin suggests using a combined iodoform/tetracycline impregnated product or TGP with MCS sealer in the treatment of teeth with long standing infections.11 The latter recommendation was taken into account when designing the study. Despite the immediate beneficial effects of MGP, a recent study found that MGP could not maintain its antimicrobial and antifungal effects after 48 and 72 hours.23

Tetracycline has been widely used in the treatment of various types of periodontal diseases that are refractory in nature and it has also been found to be effective at killing Actinobacillus actinomycetemcomitans, usually associated with aggressive periodontitis.4 Researchers such as Rosenblatt (1986) question the antimicrobial efficacy of tetracycline against common periodontic pathogens, due to the development of varying degrees of resistance to gram-positive organisms and anaerobes.24 Despite this claim, Walker et al. (1979) found tetracycline to have significant antimicrobial efficacy again
st most bacteria isolated from human periodontal pockets.25 Among these were Actinobacillus actinomycetemcomitans, Oral Campylobnacter, and Streptococcus species with minimum inhibitory concentrations in agar media under 2m/ml. More recently, Colombo et al (2003) concluded that the use of both systemic and locally delivered tetracycline significantly reduced the numbers of “red complex” bacteria for at least six months after treatment.26

Tetracycline’s ability to diffuse through and bind to dentin was clearly evident in several root specimens at the completion of the study. Figures 2A & B depict a specimen from G4 (MCS control) with distinctly yellow dentin relative to a specimen from G2 (negative control). Several studies have investigated the binding of tetracycline to dentin. The tracer molecules of the related tetracycline antibiotic dimethychlortetracycline contained in Ledermix (Lederle Pharmaceuticals, Wolfratshausen, Germany), was found to permeate through the dentin of single-rooted teeth. Both removal of the smear layer and cementum, were found to increase diffusion of the tracer molecules.27 Consequently, immediate intracanal placement of Ledermix in replanted teeth has been shown to result in statistically significantly more healing and less external resorption when compared to calcium hydroxide.28

In this study, the amount of diffused tetracycline through teeth obturated with TGP and MCS sealer was statistically insignificant relative to freestanding TGP in saline (the positive control; G3). Depending on the study, the MIC for tetracycline can be interpreted as ranging from 0.016g/ml to 2.00g/ml, as determined by Kleinfelder (1999) or from 4g/ml to 8g/ml, as recorded by Walker et al (1981).8,29 Based on the range reported by Kleinfelder (1999), teeth obturated with TGP and without MCS sealer (the MCS control; G4) and freestanding TGP (positive control; G3) had an average diffusion of tetracycline that fell within or above the MIC range; respectively. However, the higher range reported by Walker et al (1980), would mean only freestanding TGP in saline (positive control; G3) produced an average concentration of diffused tetracylcine that would be sufficiently antibacterial. Nonetheless, the amount of tetracycline diffusion from teeth obturated with TGP and MCS sealer (the experimental group; G1) fell well below both documented MIC ranges for tetracycline.

The rate of tetracycline diffusion through time decreased exponentially from 0 hours to 96 hours. These results are similar to those recorded for the rate of diffusion through dentin of dimethychlortetracycline from Ledermix used as an indirect pulp-capping agent in a study done by Abbott et al (1989) where the peak rate of diffusion was at 2 hours.29 The observed rise in tetracycline concentration from 96 hours to two weeks is likely due to its accumulation in solution from the longer time intervals chosen for this study and not due to possible reactivation of the medicated points. Such an accumulation would not be likely in vivo, where the gingival crevicular fluid is in a constant state of flux.

Based on the results of this study, the in vitro obturation of teeth with TGP and MCS sealer does not permit sufficient diffusion of tetracycline, as determined by the Ridacreen Tetracyclin, that is consistent with known minimal inhibitory concentrations.

Future research of this product should investigate whether there is some benefit to the use of activated pre-soaked TGP points as an intra-appointment medicament during root canal treatment against both endodontic and periodontal pathogens within the root canal system and the surrounding periodontium.

Dr. Mary Dabuleanu is in private practice limited to endodontics in Toronto, ON. Gabriel J. Faubert is the regional manager and senior sales manager at R-Biopharm Inc. Dr. John Molinari is the professor and chairperson of the Department of Biomedical Sciences, University of Detroit, Mercy. Dr. Frank E. Pink was formerly professor of Restorative Dentistry and associate dean office of Graduate Education and Research, chairperson for the Department of Restorative Dentistry and chairman of the Institutional Review Board, University of Detroit, Mercy. He is currently in private practice in Manistique, MI. Dr. Michael Hoen is the associate professor and Graduate Program Director, Department of Endodontics, University of Detroit, Mercy. Dr. W. Choong Foong is the associate professor, Department of Biomedical Sciences, University of Detroit, Mercy.

We would like to thank R-Biopharm, Inc. for their technical support and Medidenta International for their generous donation of Tetracycline Gutta Percha (TGP) and MCS Root Canal Sealer.

Oral Health welcomes this original article.

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14.Ridascreen tetracyclin enzyme immunoassay for the quantitative analysis of tetracycline. R-Biopharm AG; Darmstadt, Germany.

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Table 1

G1: Experimental Group

	24 hours(g/ml)	48 hours(g/ml)	96 hours(g/ml)	1 week(g/ml)	1 month(g/ml)
N of cases	15	15	15	15	15
Minimum	0.000	0.000	0.000	0.000	0.000
Maximum	0.000	0.000	0.006	0.008	0.087
Mean	0.000	0.000	0.000	0.002	0.026
Standard Dev	0.000	0.000	0.001	0.003	0.029

Table 2

G2: Negative Control

	24 hours(g/ml)	48 hours(g/ml)	96 hours(g/ml)	1 week(g/ml)	1 month(g/ml)
N of cases	3	3	3	3	3
Minimum	0.000	0.000	0.000	0.000	0.000
Maximum	0.000	0.000	0.000	0.000	0.000
Mean	0.000	0.000	0.000	0.000	0.000
Standard Dev	0.000	0.000	0.000	0.000	0.000

Table 3

G3: Positive Control

	24 hours(g/ml)	48 hours(g/ml)	96 hours(g/ml)	1 week(g/ml)	1 month(g/ml)
N of cases	3	3	3	3	3
Minimum	58.400	18.350	7.466	13.180	21.831
Maximum	85.640	24.080	15.839	14.540	30.901
Mean	68.760	22.000	10.684	13.877	27.107
Standard Dev	14.744	3.171	4.510	0.681	4.713

Table 4

G4: MCS Control

	24 hours(g/ml)	48 hours(g/ml)	96 hours(g/ml)	1 week(g/ml)	1 month(g/ml)
N of cases	3	3	3	3	3
Minimum	0.000	0.000	0.006	0.027	0.214
Maximum	0.000	0.011	0.033	0.057	0.408
Mean	0.000	0.004	0.018	0.045	0.308
Standard Dev	0.000	0.007	0.014	0.016	0.097

Table 5

Dep Var: g/ml N: 120 Multiple R: 0.985 Squared multiple R: 0.970

Source	Sum-of-Squares	df	Mean-Square	F-ratio	p-value
Group	10639.096	3	3546.365	655.580	0
Time	2056.317	4	514.079	95.033	0
Group*Time	5766.769	12	480.564	88.837	0
Error	540.951	100	5.410