Kinetics of drug release from a biodegradable local drug delivery system and its effect on Porphyromonas gingivalis isolates: An in vitro study.

BACKGROUND: Conventional anti-microbial therapy largely consisted of systemic administration of various drugs effective against periodontal pathogens, but fraught with several problems. Based on the concept of local drug delivery a bioresorbable device made of pure fibrillar collagen has been developed. The aim of this study was to study the release of Tetracycline from this collagen fiber (Type I collagen) impregnated with Tetracycline and its antibacterial activity against Porphyromonas gingivalis.
MATERIALS AND METHODS: Porphyromonas gingivalis was isolated from plaque samples of chronic periodontitis patients by using a CO2 incubator. DNA isolation was done followed by polymerase chain reaction (PCR) amplification to confirm the presence of bacteria. The release pattern of Tetracycline was assessed for a period of 10 days in water (group I) and Serum inoculated with Porphyromonas gingivalis (group II).
RESULTS: A significant presence of Tetracycline on all days in Group I and group II and the zone of inhibition was also present in both groups with a steady decline from day 1 to day 10.
CONCLUSION: Since the results were well within the therapeutic concentration of drug required to inhibit the growth of gram -ve bacteria (Porphyromonas gingivalis), this bioresorbable Tetracycline fiber has the potential for clinical application.

INTRODUCTION

The importance of bacteria in the etiology of periodontal disease and also the association of specific groups of gram –ve organisms (75%) is well established.[12] This increasing knowledge of specific bacteria as predominant agents in the development of periodontal diseases led to the use of systemic antimicrobials in the treatment of periodontitis, along with mechanical and surgical methods.[3456]

Systemic antimicrobials are, however, fraught with several problems.[7] The concept of local delivery of antimicrobials evolved as early as 1979.[8] The first slow-delivery devices were non-resorbable.[9101112]

As early as 1979, Minabe et al.[13] developed a resorbable collagen film impregnated with tetracycline which has high antibacterial activity against all periodontal pathogens[141516] and has been incorporated into a variety of devices as well.[101317181920212223] Tetracycline has been identified as having anti-collagenase effect;[2425] it inhibits the mechanisms of bone resorption,[26] tissue inflammatory activity,[27] tissue penetration, and root surface substantivity.[282930]

The critical factor in the use of a locally delivered antimicrobial agents in periodontal treatment depends upon their ability to deliver high concentrations of the drug directly within the pocket at bacteriostatic or bacteriocidal concentrations.[31]

Hence, the present study was designed to study the in vitro release pattern of tetracycline from the commercially available collagen fibers Periodontal Plus AB™ (Advanced Biotech Products Private Limited, Chennai, India) for a period of 10 days and also the antibacterial activity for the same period against a specific periodontal pathogen, Porphyromonas gingivalis.

MATERIALS AND METHODS

The material used was a bioresorbable vehicle made of type I collagen fibers, weighing 25 mg impregnated with 2 mg tetracycline hydrochloride.

Experimental design

Isolation of P. gingivalis

Genomic DNA isolation and purification

Polymerase chain reaction (PCR) amplification to confirm the presence or absence of P. gingivalis

Release pattern of tetracycline from the type-I collagen

Residual antibacterial activity of the test material.

Isolation and identification of P. gingivalis

P. gingivalis was isolated from the plaque samples of chronic periodontitis patients with a probing depth of 5 mm or more. Pooled samples from the sites with deepest probing depth were taken. Plaque samples were collected using a curette, and transported using Eppendorf tubes containing thioglycollate broth and plated in tryptic soy agar supplemented with hemin (5 μg/ml), Vitamin K (0.5 μg/ml), and menadione. This was incubated in a CO2 incubator for 24 h with 7.5% CO2 at 40°C. P. gingivalis colonies were seen after 24 h. In order to verify and isolate P. gingivalis, DNA was isolated by the standardized method as described in the Laboratory Manual of Molecular Cloning.[32] PCR amplification[32] [Figure 1] was done to confirm the presence of P. gingivalis. After isolation of P. gingivalis by PCR, the clones were subcultured by inoculating in a nutrient broth and incubated overnight in a CO2 incubator for the multiplication of the bacteria, which were used for further plating in Petri plates to study the antibacterial activity.

Figure 1

Gel electrophoresis – Single band PCR amplified product indicating the bacteria as P. gingivalis

The release pattern of tetracycline was assessed for a period of 10 days in the following:

Water

Serum inoculated with bacteria (P. gingivalis)

They were marked as follows:

Group I – Water

Group II – Serum + bacterial inoculum

Each group had 10 Eppendorf tubes.

On the initial day, 20 pieces of the test material (Tetracycline impregnated collagen fiber) were taken and placed in 20 Eppendorf tubes containing 1 ml of water and serum inoculated with P. gingivalis, and sealed with para film. These tubes were incubated at 30°C for 24 h.[11]

About 50 μl of the culture was plated on the prepared plates and the test material was placed on the initial day in these plates and incubated at 40°C in an atmosphere containing 7.5% CO2 for 24 h.[11]

On day 1, the tetracycline concentration was determined in all the samples. The test material (collagen fiber) from tube No. 1 of each group was transferred to an agar plate seeded with P. gingivalis. The liquids in other tubes were replaced fresh and again incubated at 30°C for 24 h. Thus, everyday, the number of Eppendorf tubes in each group was reduced by one until 10 days.

Tetracycline measurements

The concentration of tetracycline in the liquids was determined spectrophotometrically.[11] Tetracycline in water could be measured directly, while the serum samples had to be de-proteinized before assessment. A standard solution from a known concentration of tetracycline hydrochloride was prepared and its optical density obtained in order to determine the concentration of tetracycline present in the study sample. The standard preparation was repeated concurrently for 3 days to minimize the error in calculation. The average slope value was calculated and this was used for calculating the tetracycline concentration.

Measurement of zone of inhibition

The antibacterial activity of the test material was obtained by measuring the diameter of the zone of inhibition in centimeters around the residual test materials in the culture plate seeded with P. gingivalis, after 24 h incubation in a CO2 incubator.

Statistical analysis

Statistical package SPCC PC + (Statistical Package for Social Science, version 4.0.1) was used for statistical analysis. Mean and standard deviation were estimated from the repeat samples for each study group at different points. The mean values were compared by Student's independent t-test/Student's paired t-test/Tukey's honestly significant difference (HSD) test/analysis of variance (ANOVA). In the present study, P < 0.05 was considered as the level of significance.

RESULTS

Tetracycline concentration

This measured 379 -7 μg and was found to be statistically significant (P < 0.05) only on day 1 and 2 and not on other days [Graph 1].

Graph 1

Release pattern of tetracycline in water and serum

The concentration of tetracycline in group II was in the range of 180-548 μg. The results showed that tetracycline was present on all days with a multinodal release pattern between day 1 and day 10 [Graph 2]. Statistically the values were significantly higher on days 1, 4, 6, 7, and 9 (P < 0.05), as shown in Table 1.

Graph 2

Zone of inhibition for P. gingivalis – in vitro

Table 1

Test of significance for the mean values of absorbance of tetracycline between Group I (water) and Group II (serum+inoculums)

The difference in the mean values of tetracycline between Groups II and I was highly significant (P < 0.0001) from day 4 to day 10.

Zone of inhibition

The zone of inhibition in group I was from 4.5 ± 0.0 [Figure 2] to 0.6 ± 0.1 cm, which was statistically significant on days 1, 2, 3, and 4 than the other days (P < 0.0001).

Figure 2

Group I water – Zone of inhibition of tetracycline for P. gingivalis in culture plate on day 1

In Group II, the zone of inhibition was in the range from 4.5 ± 0 [Figure 3] to 0.9 ± 0.2 cm, which was also significantly higher on days 1, 2, 3, 4, 5, and 6 when compared to days 7, 8, 9, and 10 (P < 0.0001), as shown in Table 2.

Figure 3

Group II serum – Zone of inhibition of tetracycline for P. gingivalis in culture plate on day 1

Table 2

Test of significance for the mean values of absorbance of tetracycline between Group I (water) and Group II (serum+inoculums)

The results revealed that the collagen fiber device was able to produce substantial release of the incorporated drug over a period of 10 days in serum inoculated with bacteria, and the residual drug in the fiber also showed a zone of inhibition in the culture plates during the study period of 10 days [Figure 4].

Figure 4

Group I water, group II serum – Zone of inhibition of tetracycline for P. gingivalis in culture plate on day 10

DISCUSSION

Treatment of periodontal disease consists of the use of effective antibacterial agents as much as the use of effective therapeutic surgical and non-surgical techniques for the elimination of local factors. Local antimicrobial therapy provides for a more sustained control of the intra-pocket microbial environment and helps in maintenance during periodontal wound healing.

Various non-resorbable vehicles impregnated with tetracycline have been developed and evaluated clinically and bacteriologically for their therapeutic effect.[81021333435]

This study was aimed at studying the in vitro antimicrobial efficiency and kinetics of a collagen fiber based device impregnated with tetracycline.

The kinetics of the drug in water was seen to be a steady dissolution phenomenon that produced a homogenous reduction in the drug release rate. In serum, the rate of drug released seemed to be variable. The water and serum environment in the present study underwent a constant change on a daily basis, as the solution was exchanged daily in order to prevent drug saturation. In an effort to replicate the pathologic environment, the serum was also inoculated with an isolate of P. gingivalis.

In this study, a statistical analysis of the spectrophotometric absorbance rate revealed that the difference in the rate of drug release between water and serum with bacterial inoculums was significant, although statistical significance did not exist in the between-group data for the first 3 days. The rate of drug release during the first 3 days was almost similar in both groups and it is assumed that this is largely due to initial dissolution phenomenon. This phenomenon undergoes a steady and gradual decrease in the case of the Group I, and in Group II, an almost constant, if slightly variable, concentration of the drug is maintained.

Goodson et al., in a series of studies, have demonstrated the efficacy of tetracycline in control drug delivery devices of various types.[181920] The ideal concentration of bioavailable tetracycline should be in the range of 50-500 μg/ml.[36] As the gingival flow rate in a 5 mm pocket is in the order of magnitude 20 μl/h, a drug delivery system should be able to deliver about 0.4-2.4 mg of tetracycline for a therapeutic period of 10 days.[37] This present study also witnessed a sustained release for 10 days in serum, with a cumulative concentration of about 2000 μg/ml. The minimum concentration of the drug was 180 μg/ml and the maximum was 548 μg/ml, which was well above the therapeutic level of tetracycline required to be maintained in the gingival sulcus.

The variable pattern of release of the drug in serum could be due to the effect of bacterial collagenases and other serum products on the degradation process of the collagen fibrillar vehicle. In essence, the rate of release of the drug from the collagen vehicle, as in any resorbable control drug delivery device, is dependent on the rate of degradation of the vehicle.[2238] The degradation of collagen in a physiologic environment is vastly influenced by a variety of factors and more specifically by a group of degradation enzymes known as matrix metalloproteinases (MMPs). Several of these enzymes are tissue derived, while some of them are bacterial in origin.[31] P. gingivalis has the highest collagenolytic activity,[39] and these enzymes could potentially act on the collagen vehicle in the present study and produce a variable degradation rate.

Golub et al.,[2324] in a series of studies, established the mechanism by which tetracycline retards the collagen degradation process. It is understood that tetracycline acts as a scavenger for metal cations which are otherwise required for the action of MMPs on a collagen substrate. Golub et al. further generated clinical applications for these findings by developing chemically modified tetracyclines (CMTs) with none or little antimicrobial effect.[25] These drugs currently find vast application in the retardation of collagen degradation in the disease process, and commercial formulations of such tetracycline molecules are in use today (Periostat®).

The present study found a variation in the kinetics of drug release only in serum inoculated with bacteria, not in water. This may be the direct result of the varied rate at which the collagen vehicle was degraded, since the bacterial inoculums of P. gingivalis were also not standardized. In addition, the degradation process is further affected by the release of tetracycline itself. Consequently, it is expected that a balance between the amount of drug and the quantum of microbial insult would be maintained in proportion. This would result in degradation of the collagen vehicle, only when the microbial quantum increases as a result of diminution in the drug concentration. Sequentially, the breakdown of collagen would release in a burst release of the drug from the vehicle, which would again reduce microbial quantum until the concentration decreased below the minimum inhibitory concentration of the drug. In effect, this would result in a microbial-determined and tetracycline-determined rate of degradation and release, which would be very useful clinically, as opposed to a constant rate drug release device.

The vehicle also was able to maintain inhibitive concentrations throughout the 10-day study period in the range of 0.9-4.5 cm in serum. This was evidenced by the fact that fibrillar masses of the vehicle removed from the test environment produced a zone of inhibition in a culture plate of P. gingivalis on all days of the study with a highly significant P value (P < 0.0001).

Interestingly, the zone of inhibition in both groups was almost similar. This could mean that the collagenolytic activity and drug release in culture was constant for vehicles harvested from both groups. This provides further evidence that the rate of collagenolysis of the vehicle is the primary determination of the kinetics of drug release.

In bacterial culture, the difference in the zone of inhibition between groups was not statistically significant on any given day. Since the environment in the culture plates in both groups was identical in terms of collagenolytic effect on the vehicle and consequent drug release, the data in both groups showed a gradual and similar decrease in zone of inhibition. This suggests a gradual decrease in drug availability and residual antibacterial activity as a result of drug release in the Eppendorf tubes. With specific reference to P. gingivalis, the concentration of tetracycline required to inhibit 100% growth is in the range of 1-3.2 μM.[40] In the present study, since the zone of inhibition was present on all days in the culture plate of P. gingivalis, it is assumed that the residual drug in the fiber was within the range of inhibition for P. gingivalis.

The present study is in general agreement with few previous studies that either used the same drug or similar vehicles.[2241]

In a recent study, Periodontal Plus AB, used as a local drug delivery adjunct to scaling and root planning (SRP), in comparison to SRP alone, demonstrated mean decrease in probing depth and inflammation after 30 days and 90 days, which was statistically significant.[42]

Ruchi et al., in a comparative study of two local drug delivery systems, one containing chlorhexidine and the other containing tetracycline hydrochloride in collagen fiber as an adjunct to mechanical therapy, showed an improvement in clinical parameters.[43]

In a similar clinical study, the utilization of Periodontal Plus AB fibers demonstrated improvement in clinical as well as microbiological parameters.[44]

CONCLUSION

The collagen fiber in this study demonstrated a controlled release of the drug tetracycline for a therapeutic period in a pathologically simulated environment. This fiber is able to inhibit P. gingivalis in a culture plate for the same period of time.

Thus, it can be concluded that this novel biodegradable collagen fiber with a consistent release pattern of tetracycline in a pathologically simulated environment inhibits the growth of a primary periodontal pathogen and can be effective as an adjunct to mechanical therapy in improving the clinical parameters in chronic periodontitis.