Comparative evaluation of effects of chlorhexidine and tetracycline on neutrophil viability and functions in vitro.

BACKGROUND: Chlorhexidine (CHX) and Tetracycline (TET) are the two antimicrobial agents used in the management of periodontal infections, due to their antimicrobial potency and substantivity. The benefits and limitations of an antimicrobial agent can only be assessed by determining their relative toxicity to microbes and host cells.
OBJECTIVES: (1) To detect the effects of CHX and TET on neutrophil viability and functions in vitro. (2) To compare the effects of CHX and TET on crevicular blood neutrophils.
MATERIALS AND METHODS: Crevicular blood was collected using 5-μl pipette, stored in EDTA-containing vacutainers and sent for evaluation of Cell Viability- Neutrophils would be mixed with 0.25 volume of 0.4% trypan blue in Hank's balanced salt solution (HBSS) and counted to assess the viability. Chemotaxis- Evaluated under agarose slides, a total of 100 μl crevicular blood Neutrophils-0.2% BSA or HBSS-0.2% containing concentration of CHX and TET. Oxidative Metabolism- would be assessed using nitro blue tetrazolium. In all experiments, three concentrations of TET (0.1%, 0.05%, 0.025%) and CHX (0.01%, 0.005%, 0.0025%) respectively were used. N-Formyl methionyl leucyl phenylalanine (1 μm) in the presence of 2 μg/ml cytochalasin B will be used as the activating agent. Neutrophils would be treated with CHX and TET similarly to that for chemotaxis.
RESULTS: TET comparatively has less deleterious effects on neutrophil functions as compared with that of CHX with statistically significant results for all parameters tested.
CONCLUSION: From this study, it is inferred that CHX and TET do cause certain changes in neutrophil functions.

INTRODUCTION

Periodontitis is an inflammatory disease causing progressive destruction of the supporting structures of the tooth. Specific microorganisms have been attributed to the causation of this disease. Neutrophils are phagocytic cells that protect the host from bacterial infections and play an important role in the initiation and progression of the inflammatory response.

To fulfill this role, neutrophils adhere to activated endothelial cells, transmigrate into tissues, and migrate toward invading microorganisms. Upon arrival at an infection site, neutrophils phagocytose bacteria and kill them with reactive oxygen metabolites and microbicidal proteins. In the periodontium, they are located within gingival tissues where their concentration is greater in proximity of junctional epithelium and sulcular epithelium. Neutrophils are also present in gingival sulcus1. One area that has been poorly addressed in host pathogen relationship is the role of gingival crevicular microenvironment in periodontal disease.

Antimicrobial treatment of periodontitis aims at eradicating or controlling specific pathogens. Prime candidates for antibiotic therapy are patients with recently diagnosed active periodontitis or a history of recurrent disease who fail to stabilize following mechanical or surgical therapy.

Chlorhexidine (CHX) and tetracycline (TET) are two antimicrobial agents frequently used in the management of periodontal infections, due to their antibacterial potency and substantivity.[1] The benefits and limitations of an antimicrobial agent can only be assessed by determining their relative toxicity not only to microbes but also host cells.[2]

As neutrophils sense as the first line of host defense against microbial infections, it is needed to detect if CHX or TET cause any changes in the functions of crevicular blood neutrophils, in addition to their antimicrobial activity, under identical experimental conditions. The hypothesis addressed by this study was that CHX and TET could alter the viability and functions of neutrophils.[34] This study addresses this hypothesis by comparative evaluation of effects of CHX and TET on neutrophils". This addressal is carried out through comparative evaluation of effects of CHX and TET on neutrophil viability and functions in vitro.

Objectives

To detect the effects of CHX and TET on neutrophil viability and functions in vitro.

To compare the effects of CHX and TET on crevicular blood neutrophils.

MATERIALS AND METHODS

One ml of crevicular blood was collected using 5-μl capillary pipettes and stored in vacutainers containing EDTA.

The criteria for selection of subjects were as follows

Inclusion criteria

Adult periodontitis patients

Patients above 35 to 50 years of age

Sample size: 30 patients

Pocket probing depth >4 mm

Exclusion criteria

Subjects with chronic systemic illness or using episodic medication such as antibiotics, anti-inflammatory, or antiepileptic drugs

Subjects with established neutrophil dysfunctions

Subjects using tobacco in any form

Aggressive periodontitis cases

Pregnant and lactating mothers

Subjects already using products containing CHX or TET.

Procedure

Neutrophil isolation and purification was done by dextran sedimentation technique and Hypaque-Ficoll gradient centrifugation procedure and each sample was divided into 3 groups for baseline, TET, and CHX and evaluated for three different concentrations of TET and CHX.

Hank's balanced salt solution (HBSS)

N-Formyl methionyl leucyl phenylalanine (FMLP)

CHX and TET at a concentration of 10Mm made in water, absolute alcohol, and 0.85% NaCl, subsequently diluted in HBSS 0.2% BSA.

Hypaque: Sodium diatrizoate

Ficoll

Order T400

Weigh out 32 g and dissolve by heating in 250 ml of distilled H 2 O

Store at 40°C

Hypaque - Ficoll solution

Add two vials of hypaque to 250 ml Ficoll solution and bring up to 500 ml/distilled H2 O. Filter through 0.45 m filter to remove bacteria.

Final concentration of Hypaque is 9.97% wt/vol and that of Ficoll is 6.35%, specific gravity is 1.08.

HBSS without Ca2+ and Mg2+

Cell viability

Neutrophils treated with the test reagent were mixed with 0.25 vol of 0.4% Trypan blue in HBSS and counted immediately to assess the viability by Trypan blue exclusion dye. At least 3 microscopic fields of approximately 100 neutrophils were enumerated for each test [Figure 1].

Figure 1

Cell viability

Chemotaxis

Equipment

37°C incubator (moist and 5% CO2)

A means for low-power magnification: a micro-projector [Tri-Simplex (Bausch and Lomb, Rochester, NY] enlarger or Optomax Image Analyzer interfaced with a TV monitor (CPU-2, Micromeasurements, Cambridge, England) was used.

Reagents

0.024 g/ml agarose

Litex-type, HAS, Accurate Chemical and Scientific Corp., (Hicksville, NY).

Dissolve 0.24 g/10 ml sterile dH 2 O by heating in boiling water for 10 to 15 minutes.

Cool to 48°C

Supplemented MEM - keep warm at 48°C

2.0 ml 10x MEM

2.0 ml heat-inactivated pooled human serum

0.2 ml 7.5% sodium bicarbonate

5.8 ml sterile dH 2 O

Preparation of agarose for 60- × -15-mm tissue culture plates

Add 10 ml prewarmed supplemented MEM to 10 ml 0.024 g/ml agarose.

Add 5.0 ml mixture to each culture plate (#3002 Falcon Plastics, Oxnard, CA).

Allow to solidify

See below for making wells in agarose.

10-8 M fmet-leu-phe as chemoattractant

Staining reagents per plate:

3 to 5 ml methanol

3 to 5 ml formalin

Wright's stain

MEM with 2mM L-glutamine and 100 U/ml penicillin and 100 g/ml streptomycin.

For chemotaxis

Add 0.01 ml 2.5 × 10 7 cells/ml to B well (center)

Add 0.01 ml chemoattractant to A well

Add 0.01 ml buffer to C well

For chemokinesis

Add 0.01 ml 2.5 ×10 7 cells plus chemoattractant to A + B well.

Add 0.01 ml chemoattractant to both A wells.

Incubate PMNs at 37°C in humidified 5% CO 2 incubator for 2 hours (monocytes for 10 to 18 hours).

After incubation, fix and stain cells with Wright's stain.

Calculations

Project plates onto white background so that the well is magnified to 5 cm (not done when using image analyzer to project onto TV monitor) [Figure 2].

Figure 2

Chemotaxis

Chemotaxis = linear distance (in cm) that cells (from B) have migrated from margin of well toward chemoattractant (A).

Chemokinesis = linear distance that cells have migrated from margin of well A + B toward both A wells.[34]

Nitro blue tetrazolium test

Principle

This is a screening test which measures the oxidative burst. Nitro Blue Tetrazolium is an electron acceptor used to detect indirectly the production of superoxide by stimulated PMNs as OXIDATIVE BURST—outlined in the following equation:

NBT (yellow) + O 2 - → Formazan (black) + O2

The nitro blue tetrazolium (NBT) slide test provides an easy method to screen PMNs for the capacity to undergo oxidative metabolism. PMNs that fail to reduce NBT include those with Chronic Granulomatous Disease (no oxidative activity), glucose 6-phosphate deficiency (NADPH store rapidly depleted), etc.

Procedure

200 μl of HBSS was taken in each of the four test vials. The pH was adjusted to 6.7, 7.2, 7.7, and 8.2, respectively. Three aliquots of 50 μl of NBT, 50 l of endotoxin, and 100 μl of whole blood were added to each of the test vials. These were incubated for 20 minutes at 37°C, then kept as such for another 20 minutes at room temperature. Smears were prepared and allowed to dry. The smears were fixed and stained with Giemsa [Figure 3].

Figure 3

Oxidative burst

Statistical analysis

It was performed using the students paired “t-test” and unpaired “t-test.”

RESULTS

Observations

The results show the values obtained for three parameters of cell viability, chemotaxis, and oxidative burst at three different concentrations of CHX (0.01%, 0.005%, and 0.0025%) and TET (0.1, 0.05, and 0.025%)

Table 1 and Figure 4 show the effects of CHX and TET on PMN viability at the three concentrations. TET at 0.025% conc. rendered the cells greater viability of PMNs, i.e., 76.86 cells, whereas 0.05% conc. of TET about 72.30 neutrophils and 69.13 neutrophils at 0.005% conc., respectively.

Table 1

Cell viability

Figure 4

Mean values obtained for cell viability

CHX at all concentrations shows less viable cells of about 62.700 PMNs for 0.0025% conc., 55.833 for 0.005% conc., followed by 50.433 for 0.01% concentration, depicting that lesser concentration attribute to the greater viability of cells.

Table 2 and Figure 5 show chemotaxis, the migration of neutrophils in micrometers. The mean values of migration are maximum for TET of 2.2033 μm migration for 0.025% conc., followed by 0.05% conc. of about 1.5933 μm, and finally 0.01% conc. showed just a mean migration of 0.97.

Table 2

Chemotaxis

Figure 5

The mean values obtained for chemotaxis

However, CHX exhibited mean migration of 1.667 μm at 0.0025% conc., up to 0.97 μm at 0.005% conc., and 0.4133 μm at 0.001% conc.; lesser the concentration, more migration these neutrophils had shown.

Table 3 and Figure 6 show the mean values for oxidative burst, along with their baseline values. TET at 0.1% conc. showed 10.033 cells; 0.05% conc., 11.86 cells; and 0.025% conc., 14.26 cells.

Table 3

Oxidative burst

Figure 6

The mean values obtained for oxidative burst

However, CHX 0.01% conc. shows oxidative burst 27.033 cells, 0.005% conc. of 28.56 cells, and 0.0025% conc. of 32.9 cells; the lesser the concentration, more number of cells showed oxidative burst.

Table 4 compares the effect of different concentration of TET on PMN's viability, when at concentration of 0.01% conc. - 0.05% conc. (P=1.3741), 0.05% conc. - 0.025% conc. (P=1.247), and 0.025%- 0.1%, (P=1.758) which are all highly statistically significant.

Table 4

Comparison between different concentrations of Tetracycline - Cell viability

Table 5 shows values that were obtained for CHX, which were also highly significant.

Table 5

Comparison between different concentrations of Chlorhexidine - Cell viability

Table 6 represents the values obtained for comparison between different concentration of TET for chemotaxis; maximum values were obtained for the concentration of 0.05-0.025% conc. (P=3.571), which is also highly significant.

Table 6

Comparison between different concentrations of Tetracycline - Chemotaxis

Table 7 represents values after comparison of different concentration of CHX for chemotaxis with P=1.1221 for 0.005-0.0025% and here too, all the values were highly significant.

Table 7

Comparison between different concentrations of Chlorhexidine - Chemotaxis

Table 8 shows the comparison of NBT test results that were carried out for different concentrations of TET; the results were highly significant except for 0.1% conc.- 0.05% conc. (1.1399), which was not very significant.

Table 8

Comparison between different concentrations of Tetracycline - Nitro blue tetrazolium

Table 9 shows the values obtained for CHX, with all the values being statistically significant.

Table 9

Comparison between different concentrations of Chlorhexidine - Nitro blue tetrazolium

DISCUSSION

The benefits and limitations of TET and CHX antimicrobials can only be assessed by their relative changes to the microbes as well as changes in the host cells.[56]

This study evaluated the effects of CHX and TET on neutrophils which are the first line of host defense. Functions of neutrophils were assessed under three concentrations of TET (TET 0.1%, 0.05%, and 0.025%) and CHX (0.01%, 0.005%, and 0.0025%). CHX was taken at a lesser concentration of TET, as CHX at a similar concentration of TET in pilot study proved to be toxic to the neutrophils and the parameters could not be tested. Investigators in many studies have used 0.02% to 0.06% conc. of CHX as an irrigant, totaling up to 120 mg/day, therefore suggesting that it would require a larger dose than rinsing. The concentrations taken in this study was sufficient enough for the parameters to be tested.[78]

For cell viability, the number of viable cells was counted in the present study. Microscopic methods, as utilized in the present study, to quantitate cell association require prolonged and tedious counting; therefore, at least three microscopic fields of approximately 100 neutrophils were enumerated for each test.[9]

The results as depicted in Figure 4 for cell viability shows the bar longest in relation to TET at the concentration of 0.025% of 76.866 cells and 62.7 cells with 0.0025% concentration of CHX. Followed by 0.05% concentration of about 72.3 cells for TET and 58.3 cells for CHX, finally 0.01% concentration rendered only 69.13 cells for TET and for CHX was just 50.43 cells. Thus stating that lesser the concentration used, the more number of cells were viable, and also shows that TET rendered more cells viable compared to CHX with P<2.01092. Another parameter that was evaluated was chemotaxis, which was done using agarose-coated slides. Since neutrophils kill organisms by generation of reactive oxygen species,[10] the ability of these two drugs to synergize or inhibit FMLP-induced superoxide anion generation was examined. Figure 5 presents the mean values that were obtained for chemotaxis showing that neutrophils underwent a greater chemotaxis of 2.2033 μm at 0.025% conc. for TET, followed by 1.533 μm for 0.05% conc., and 0.97 μm at conc. 0.1%. The neutrophils showed least chemotaxis to CHX at 0.01% with migration of just 0.4133 μm, followed by 0.5191 μm for 0.0025% conc., and finally 0.9767 μm for 0.005% conc. This meant greater the concentration, lesser the migration of neutrophils.

The third parameter to be assessed was oxidative burst; as seen in Figure 6, CHX for the concentration of 0.0025% up to 14.26 cells, followed by 11.86 cells for 0.005% conc., and up to 10.033 cells for conc. 0.01%., whereas TET even at a highest concentration of 0.01% showed 27.03 cells. From this study, it is inferred that CHX and TET do cause certain changes in the neutrophil functions with CHX having more damaging effects compared with TET.[1112]

Normal neutrophil functions vary on a variety of receptors, enzymes, and regulatory proteins that are sensitive to changes in pH.[13] Outside a certain range of pH, i.e., 7.0 to 7.5, changes on ionizable amino acid residues result in modification of the tertiary structure of the protein and eventually lead to denaturation. The optimum pH is a characteristic more of the enzyme than of the particular substrate.[14]

The results of the present study correlated with the study that checked for suppression of human neutrophil functions by TETs, in which the authors concluded the study stating that TETs may suppress neutrophil-mediated tissue damage by inhibiting their migration and degranulation and potentially more importantly, by suppressing synthesis of oxygen radicals.[14]

The effects of CHX on neutrophil functions were due to its ability to disrupt the cell membranes. CHX induced rapid lysis of neutrophils and release of granule enzymes at similar concentrations, suggesting that the inhibition of chemotaxis and oxidative burst may be due to cytolysis rather than inhibition of specific cellular functions. It is of interest that CHX neither dismutates superoxide anions nor inhibits lysozyme function. CHX is also a long-acting surfactant and is not inactivated in solution for at least 1 hour. In addition to neutrophils, CHX has shown to cause alterations in epithelial cells and macrophages.[15]

The MIC of CHX for periodontal microbes is shown to be 0.0008% - 0.05%. The MIC for microbes and concentrations of CHX which are inhibitory to neutrophil functions are within the range; CHX can induce degranulation in neutrophils at concentrations that are sufficient to kill only the most sensitive microbes.[14]

Abundant neutrophils have been found within the connective tissue, junctional epithelium, and gingival crevice (Schweder 1970, Attrtom 1971, Lehner 1976).[15] It now appears likely that neutrophils serve a protective function. Although evidence exists to suggest that systemic neutrophil disorders are associated with rapid periodontal breakdown, it seems unlikely that such systemic defects occur with enough frequency in the population to account for the prevalence of all rapidly progressive periodontitis.

CONCLUSION

CHX and TET are the most widely prescribed antimicrobials commonly used as an adjunct in periodontal therapy; however, the findings in the present study clearly illustrates that these antimicrobial agents do cause alterations in neutrophil functions posing a threat to neutrophil defense activity, and on comparison TET proves to have a less deleterious effects on neutrophil activity compared with CHX.

The gold standard efficacy and substantivity of CHX cannot be ignored, but the results of the present study clearly shows certain disadvantageous effects of CHX toward neutrophil functions, and on comparison TET could prove to be safer and might be of better therapeutic value in patients with known neutrophil defects, bearing in mind that these changes in neutrophils might cause changes in tissue pathology and may affect wound healing.

Since the efficacy of this drug can be drastically improved by controlling the local concentrations of the drug, further research experimentation in the area of controlled drug delivery systems might be beneficial in optimizing the use of these agents in therapeutic regimens. However, further studies are still required to test and confirm the accurate effects of these two drugs and the effectiveness of these antimicrobials also needs to be evaluated in in vivo trials.