cytotoxicity

1

TITLE PAGE

Title of the article: Comparative evaluation of the cytotoxicity of MTAD, 2% chlorhexidine and 5.25% sodium hypochlorite on human red blood corpuscles.

Contributors

1. Dr. Krishna Prasad Shetty MDS

Professor and Head

Department of Conservative dentistry and Endodontics

Navodaya Dental College & Hospital. Raichur

Karnataka. India

Email: [email protected]

2. Dr. Sarvepalli Venkata Satish MDS

Professor



Karnataka. India


3. Dr. Krishnarao Kilaru MDS, DNB

Reader



Karnataka. India.


4. Dr. Ratnakar P. MDS

Reader


H K E S Dental College. Gulbarga

Karnataka. India

2


5. Dr. Vijay Reddy Venumuddala MDS

Professor and Head

Hitech Dental College and Hospital. Bhubneswar

Orissa. India


6. Dr. Kalyana Chakravarthi Ponangi BDS

Post Graduate Student.



Karnataka. India


Department and institution

Department of Conservative dentistry and Endodontics.


Karnataka. India

Running title: cytotoxicity of three endodontic irrigants on human red blood corpuscles:

MTAD, 2% chlorhexidine and 5.25% sodium hypochlorite.

Key-words: Cytotoxicity-Endodontic irrigants; Red Blood Corpuscles; MTAD-

cytotoxicity; Chlorhexidine-cytotoxicity; Sodium Hypochlorite-cytotoxicity.

3

Corresponding Author:

Name:Dr. Krishna Prasad Shetty

Address: Department of Conservative dentistry and Endodontics

Navodaya dental college & hospital. Navodaya nagar.

Mantralayam road. Raichur. PIN – 584103

Karnataka. India.

Phone numbers: 919611282811

Facsimile numbers: 08532-223043

E-mail address: [email protected]

mailto:[email protected]

4

Title of the article: Comparative evaluation of the cytotoxicity of 5.25% Sodium

hypochlorite, 2% Chlorhexidine & MTAD on human red blood corpuscles: An in-vitro

study

Abstract:

Aim: The aim of this study was to analyse the cytotoxicity of various volumes of 5.25%

Sodium hypochlorite, 2% Chlorhexidine gluconate and MTAD by checking for

haemolysis of human red blood corpuscles.

Methodology: Hundred micro litres of diluted Red Blood Corpuscles obtained through

centrifugation was added to three groups (Group I: 5.25% Sodium hypochlorite, Group

II: 2% Chlorhexidine, Group III: MTAD) of six test tubes each. Individual irrigants per

group were added in increasing volume starting from the second test tube keeping the

first one as the control. After incubation for three minutes haemoglobin content was

measured using an automated haemoanalyser. Data was analyzed using one sample t

test.

Results: Sodium hypochlorite is the most cytotoxic followed by MTAD and

chlorhexidine the least.

Conclusions: This study suggests that these irrigating fluids do cause detrimental

effects on vital tissues. Sodium hypochlorite though an irrigant of choice should be used

cautiously. Chlorhexidine should be used when excessive extrusion is expected. MTAD

5

is the irrigant of choice because of its antimicrobial efficacy and smear layer removing

ability.

Key Messages: 2% Chlorhexidine is less cytotoxic when compared to 5.25% Sodium

Hypochlorite and MTAD. Therefore 2% Chlorhexidine is the irriganting solution of choice

in teeth with incomplete root development and perforations.

6

Introduction:

Successful endodontic treatment depends on the ability of the operator to mechanically

and chemically clean the root canal system and subsequently obturate it three

dimensionally (1). Endodontic therapy is primarily based on the removal of potentially

noxious stimuli from the complex root canal system (2).

The complexity of the root canal system is, however, one of the determining factors in

the failure of root canal treatment even in properly treated teeth, due to irregular and

sometimes unpredictable spaces that characterize root canals (3, 4). The persistence of

residual pulp tissue, infected dentin or bacteria in the root canal system is responsible

for treatment failure (5). Irrigation, therefore, plays a crucial role in determining the

outcome of root canal treatment (6). Irrigants flush debris from canals and assist in

reducing microbial flora of infected canals and help to dissolve the necrotic tissues.

An endodontic irrigant should be non toxic when it comes in contact with vital tissues

and non caustic to the periodontal tissues (7). A potential complication of irrigation is the

forced extrusion of the irrigant and debris through the apex. Tissue cytotoxicity is

therefore a major concern when choosing an endodontic irrigant for root canal

treatment.

Sodium hypochlorite has solvent activity for both necrotic and vital tissues (2). It is

known in medical fields since the beginning of 20th century and was introduced as a part

Text

7

of endodontic treatment in 1936 by Walker. Literature is full of studies showing severe

irritations when concentrated solution were inadvertently forced into the periapical

tissues beyond the apex of the tooth under endodontic treatment (8, 9).

Chlorhexidine was developed in the late 1940s in the research laboratories of Imperial

Chemical Industries Ltd (7). Chlorhexidine is a cationic biguanide that possesses broad

antibacterial activity in combination with relatively low toxicity and the ability to bind to

skin and mucous membranes.

MTAD was introduced as an alternative to EDTA to remove the smear layer by

Torabinejad et al. It is a mixture of 3% doxycycline, 4.25% citric acid and detergent-

Tween 80. It has a combined chelating and antibacterial property (10).

So the main objective of this study is to compare the cytotoxicity of 5.25%sodium

hypochlorite, 2%chlorhexidine and MTAD.

8

Materials and Methods:

The informed consent of all human subjects who participated in the experimental

investigation reported or described in this manuscript was obtained after the nature of

the procedure and possible discomforts and risks had been fully explained. And the

institutional IRB approved the protocol.

Red Blood Corpuscles are chosen as the convenient cell type as they are readily

available and whose intracellular haemoglobin content can be easily measured. Fresh

blood was drawn from a human volunteer and collected in EDTA bottles. Centrifugation

of the blood is done, plasma removed and packed cell volume of Red Blood Corpuscles

is obtained. Red Blood Corpuscles are washed with saline and centrifuged several

times to remove white cells and any traces of plasma. One millilitre of packed cell

volume is added to four millilitres of saline to increase the volume of blood to five

millilitres. Hundred micro litres of this diluted Red Blood Corpuscles is added to 18 test

tubes with six test tubes in each group. The groups are as mentioned below.

Group I: 5.25% Sodium hypochlorite (VENSONS INDIA)

Group II: 2% Chlorhexidine gluconate (RC-CHLOR, DEOR, INDIA)

Group III: MTAD (DENTSPLY Tulsa Dental, United States)

For all the three groups, the first test tube is kept as a control in which no irrigant is

added. In the second test tube ten micro litres of the irrigant is added. Twenty micro

litres is added to third test tube, thirty micro litres to the fourth test tube, forty micro litres

to the fifth test tube and fifty micro litres to the sixth test tube.

9

After an incubation time of three minutes, haemoglobin percentage after haemolysis of

red blood corpuscles is noted using an automated haemoanalyser (ABX MICROS 60).

The haemoanalyser measures the haemoglobin content of the remaining red blood

corpuscles after haemolysis. For all the three groups the experiment is repeated three

times and the mean value is taken.

Data obtained in the present study is subjected to statistical analysis using one sample t

test. A p value of less than .05 is considered as significant. Statistical analysis is done

using Minitab V.14 software.

10

Results:

Group I (5.25% Sodium Hypochlorite) showed significant cytotoxicity in comparison to

the control group. The cytotoxicity of the irrigant is directly proportional to the increase

in the volume and the exposure time. At a volume of fifty micro litres, Sodium

Hypochlorite showed complete haemolysis of the Red Blood Corpuscles as shown in

Table 1.

GROUP I – 5.25% Sodium HypochloriteVolume of

the irrigant in

micro litres

Percentage of haemoglobin

Trial I


Trial II


Trial III

0(control) 4.4 4.4 4.410 1.9 2.0 2.020 1.0 0.9 1.130 0.3 0.4 0.340 0.1 0.2 0.150 0 0 0

Table 1: Sodium Hypochlorite shows complete haemolysis at a volume of 50 micro

litres.

Group II (2% Chlorhexidine gluconate), regarded as the safest among the three irrigants

showed cytotoxicity which is statistically significant. The cytotoxicity is least when

compared to MTAD and Sodium Hypochlorite. The test results are shown in Table 2.

11

GROUP II – 2% Chlorhexidine gluconateVolume of irrigant in

micro litres

Percentage ofhaemoglobin

Trial I


Trial II


Trial III0(control) 4.4 4.4 4.4

10 3.7 3.5 3.720 3.5 3.5 3.430 3.3 3.3 3.240 3.0 2.8 3.050 2.8 2.6 2.8

Table 2: Reduction in the haemoglobin percentage as the irrigant volume increases

Group III (MTAD) showed cytotoxicity which is statistically significant but lesser when

compared to sodium hypochlorite and more when compared to chlorhexidine. The test

results are shown in Table 3.

GROUP III – MTADVolume of irrigant in

micro litres


Trial I


Trial II


Trial III0(control) 4.4 4.4 4.4

10 3.5 3.6 3.620 3.5 3.4 3.430 3.1 3.2 3.040 2.9 3.0 2.950 2.7 2.6 2.7

Table 3: Reduction in the haemoglobin percentage as the irrigant volume increases.

Chlorhexidine is the least cytotoxic of the compared irrigants.

The mean and standard deviation of the percentage of haemoglobin for all the three

irrigants using one sample t test, depicting significance is shown in Tables 4, 5 and 6.

12

Group I - 5.25% Sodium Hypochlorite

Volume of the irrigant

in micro litres

Min MaxMean ± SD

Mean differe

nce

95% CI differenc

e

t-value

p-value Remarks

10 1.9 2.01.97

± 0.06

2.432.29 – 2.57

73P<0.000

2Significanc

e

20 0.9 1.11 ± 0.1

3.403.15 – 3.65

58.89

P<0.0003

Significance

30 0.3 0.40.33

± 0.06

4.073.92 – 4.21

122P<0.000

1Significanc

e

40 0.1 0.20.13

± 0.06

4.274.12 – 4.41

128P<0.000

1Significanc

e

Table 4: Statistically significant cytotoxicity shown by sodium hypochlorite at all the

volumes tested.

13

Group II - 2% Chlorhexidine gluconate

Volume of the irrigant

in micro litres

Min MaxMean ± SD

Mean differenc

e

95% CI differenc

e

t-value

p-value Remarks

10 3.5 3.73.63

± 0.11

0.760.47 - 1.05

11.5 P<0.007 Significance

20 3.4 3.53.47

± 0.06

0.930.79 – 1.07

28 P<0.001 Significance

30 3.2 3.33.27

± 0.06

1.130.99 – 1.27


40 2.8 3.02.93

± 0.11

1.471.18 – 1.75


50 2.6 2.82.73

± 0.11

1.671.38 – 1.95


Table 5: Statistically significant cytotoxicity shown by chlorhexidine gluconate at all the

volumes tested.

14

Group III – MTAD

Volume of the

irrigant in

micro litres

Min MaxMean ± SD

Mean differenc

e

95% CI differenc

e

t-value

p-value Remarks

10 3.5 3.63.57

± 0.06

0.830.68 – 0.97


20 3.4 3.53.43

± 0.06

0.970.82 – 1.11


30 3.0 3.23.1 ± 0.06

1.301.05 – 1.55

22.52 P<0.002 Significance

40 2.9 3.02.93

± 0.06

1.471.32 – 1.61


50 2.6 2.72.63

± 0.06

1.771.62 – 1.91


Table 6: Statistically significant cytotoxicity shown by MTAD at all the volumes tested.

Graphical representation of the fall in the percentage of haemoglobin with the increase

in the volume of the irrigant for all the three groups is shown in Graph 1.

15

Discussion:

Sodium hypochlorite is considered as an ideal irrigant in endodontics (Grossman et al.,

1941) (11). Hypochlorite preparations are sporicidal, virucidal and show far greater

tissue dissolving effects on necrotic than on vital tissues. These features prompted the

use of aqueous sodium hypochlorite in endodontics as the main irrigant as early as

1920 (12).

Various concentrations of sodium hypochlorite from 0.5% to 5.25% have been tried out

(13, 14). Higher the concentration better will be the antimicrobial effect and the tissue

dissolving capacity. At the same time higher concentration also carries the risk of

toxicity and tissue reaction. It is shown that 1% sodium hypochlorite is sufficient to

dissolve the pulp tissue (15). Higher concentrations of sodium hypochlorite are proven

to have greater bactericidal effect (16). Sodium hypochlorite (5.25%) in 1:1000 dilution

caused complete hemolysis of Red blood cells in vitro (Pashley et al., 1985) (17). On

investigating various concentrations of Sodium Hypochlorite (0.25%, 0.025 % and

0.0125%) for antimicrobial activity and tissue toxicity at varying time intervals it is found

that 0.025% of Sodium Hypochlorite is bactericidal and not tissue toxic and 0.25%

concentration of Sodium Hypochlorite showed tissue toxicity (Heggers et al., 1991)

(18).Sodium Hypochlorite in a concentration of 0.55% induced an inflammatory

response (M.Tanomaru Filho et al., 2002) (19). Sodium hypochlorite is toxic enough to

cause complete haemolysis according to this study.

16

Chlorhexidine is recommended as an endodontic irrigant because of its antimicrobial

activity, presumed non toxicity and intracanal substantivity (20). At higher

concentrations, Chlorhexidine results in extensive cell damage, coagulation of

cytoplasm, and precipitation of proteins and nucleic acids (21).The concentration often

used in endodontic therapy is 2% as it is more effective in less time when compared to

other concentrations of Chlorhexidine ranging from 0.002% to 2% (22).

Chlorhexidine is toxic to human gingival cells and the toxic potency is dependent on the

length of exposure and the composition of the exposure medium (23). On comparing the

inflammatory response of 0.5% Sodium Hypochlorite, 2% Chlorhexidine digluconate,

and phosphate buffered saline, it is found that 2% Chlorhexidine injection is similar to

the phosphate-buffered saline control at all times tested, while the 0.5% Sodium

Hypochlorite injection resulted in significant inflammation (Tanamaru Filho et al., 2002)

(20).On treating fifty pieces of bovine pulp tissue with normal saline, MTAD, 2%

Chlorhexidine digluconate and 2.5% Sodium Hypochlorite it is found that Chlorhexidine

has the weakest tissue dissolution capacity(Khademi et al., 2007) (24).

Chlorhexidine is recommended as an alternative in patients allergic to sodium

hypochlorite or in teeth with incomplete root formation (22) and crestal perforations to

prevent inflammatory response in proximity to the epithelial attachment (25).

Bio PureMTAD (Dentsply, Tulsa, OK) is designed to be used as a final root canal rinse

before obturation (11). Tetracycline has many unique properties of low pH and acts as a

17

calcium chelator and cause enamel and root surface demineralization (26). MTAD is

effective in removing the smear layer along the whole length of the root canal and does

not produce any signs of erosion or physical changes in dentin (27, 28 and 29). MTAD

is effective against Enterococcus faecalis, and it is less cytotoxic than a range of

endodontic medicaments, including eugenol, hydrogen peroxide (3%), EDTA, and

calcium hydroxide paste (30). In this study MTAD is found to be less cytotoxic than

sodium hypochlorite and more cytotoxic than chlorhexidine.

Acknowledgement

We affirm that we have no financial affiliation or involvement with any commercial

organization with direct financial interest in the subject or materials discussed in this

manuscript, nor have any such arrangements existed in the past three years. We deny

any conflict of interest related to this study.

18

Conclusion:

The ideal functions of an irrigant are to dissolve both the organic as well as inorganic

components, render the root canal free of microbes and be non toxic to the tissues. No

single irrigant accomplishes all the functions.

This study suggests that these irrigating fluids do cause detrimental effects on vital

tissues. The clinical situation, concentration used, exposure time to the agent and the

exposure surface area are important factors which affect the cytotoxicity of the irrigating

solution. Therefore taking into account the limitations & merits of the irrigating solutions,

if used judiciously, it adds to the success of root canal therapy.

19

References:

1. Schilder H. Cleaning and shaping the root canal. Dental Clinics of North

America 1974; 18:269-96.

2. Cemil Yesilsoy, Eugene Whitaker, Deborah Cleveland, Eric Phillips and Martin

Trope. Antimicrobial and toxic effects of established and potential root canal

irrigants. Journal of Endodontics 1995; 20:513-5.

3. Nair PN. On the causes of the persistent apical periodontitis. A review.

International Endodontic Journal 2006; 39:249-81.

4. Gulabivala K, Patel B, Evans G. Effects of mechanical and chemical

procedures on root canal surfaces. Endodontic Topics 2005; 10:103–22.

5. Moorer WR, Wesselink PR. Factors promoting the tissue dissolving capability

of sodium hypochlorite. International Endodontic Journal 1982; 15:187–96.

6. Filippo Santarcangelo. Root Canal Irrigation in Modern Endodontics: From

routine to complex Part One: The Routine [WWW document]. URL

http://en.zerodonto.com/2012/10/root-canal-irrigation-in-modern.html

[Accessed on 27 Nov 2012].

7. M.Zehnder. Root Canal Irrigants. Journal of Endodontics 2006; 32:389-98.

8. Witton R, Henthorn K, Ethunandan M, et al. Neurological complications

following extrusion of sodium hypochlorite solution during root canal treatment.


20

9. Gursoy UK, Bostanci V, Kosger HH. Palatal mucosa necrosis because of

accidental sodium hypochlorite injection instead of anesthetic solution.


10.Torabinejad M, Khademi A A, Babagoli j, Cho Y, Johnson W B, Bozhilov K

(2003) A new solution for removal of smear layer. Journal of Endodontics 2003;

29:170-5.

11.Grossman L I, Meiman B W (1941) Solution of pulp tissue by chemical agents.

Journal of American Dental Association 1941; 28:223-5.

12.Kandaswamy D, Venkateshbabu N. Root canal irrigants. Journal of

Conservative Dentistry 2010; 13:256-64.

13.Gomes BPFA, Ferraz CCR, Vianna MEVB, Berber FB. In vitro antimicrobial

activity of several concentrations of sodium hypochlorite and Chlorhexidine

Gluconate in the elimination of Enterococcus Faecalis. International

Endododontic Journal 2001; 34:24-8.

14.M E Vianna, BPFA Gomes. In vitro evaluation of the antimicrobial activity of

Chlorhexidine and sodium hypochlorite. Oral surgery Oral Medicine Oral

Pathology 2004; 97:79-84.

15.Sirtes G, Waltimo T, Schaetzle M, Zehnder M. The effect of temperature on

sodium hypochlorite short-term stability, pulp dissolution capacity, and

antimicrobial efficacy. Journal of Endodontics 2005; 31: 669-71.

16.Arieh Y Kaufman, Senia Keila. Hypersensitivity to sodium hypochlorite. Journal

of Endodontics 1989; 15:224-6.

21

17.EL Pashley, N.L Birdsong, k Bowman and D.H pashley. Cytotoxic effects of

Sodium hypochlorite on vital tissue. Journal of Endodontics 1985; 11:525-8.

18. Heggers JP, Sazy A J, Stenberg BD, Strock LL, Mc Cauley RL, Hernon DN,

Robson MC. Bacterial and wound healing properties of sodium hypochlorite

solutions. Journal of Burn Care Rehabilitation 1991; 12:420-4.

19.M Tanomaru Filho MR Leonardo L .A .B Silva, F .F Anibal and L.H Faccioli.

Inflammatory response to different endotoxic irrigating solutions. International

Endodontic Journal 2002; 35:735-9.

20.Leonard MR, Tanomaru Filho, M.Silva CAB, Nelson Filho N, Bonifacio KC. In

vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating

solution. Journal of Endodontics 1999; 25:167-71.

21.M E Vianna, BPFA Gomes. In vitro evaluation of the antimicrobial activity of

Chlorhexidine and sodium hypochlorite. Oral surgery Oral Medicine Oral

Pathology 2004; 97:79-84.

22.Lessa, F C Rosetti, Nogueira,F Silveira. Direct and transdentinal antibacterial

activity of chlorhexidine. American Journal of Dentistry 2010; 25:255-9.

23.Babich H, Wurzburger BJ, Rubin Y L, Sinensky MC, Blau L (1995) An in Vitro

study on the cytotoxicity of Chlorhexidine digluconate to human gingival cells.

Cell Biol Toxicol 1995; 11:79-88.

24.Abbasali Khademi, Ehsan usefian, Mahboobe F. Tissue dissolving ability of

several endodontic irrigants on bovine pulp tissue. Iranian Endodontic Journal

2007; 2:65-67.

22

25.Fuss Z, Trope M. Root perforations classification and treatment choices based

on prognostic factors. Endodontics and Dental Traumatology 1996; 12: 225-64.

26.K. Bjorvatn, N. Skaug, and K. A. Selvig. Tetracycline-impregnated enamel and

dentin: duration of antimicrobial capacity. Scandinavian Journal of Dental

Research 1985; 93:192–7.

27.M. Torabinejad, Y. Cho, A. A. Khademi, L. K. Bakland, S. Shabahang. The

effect of various concentrations of sodium hypochlorite on the ability of MTAD

to remove the smear layer. Journal of Endoddontics 2003; 29:233–9.

28.R. E. Beltz, M. Torabinejad, M. Pouresmail. Quantitative analysis of the

solubilizing action of MTAD sodium hypochlorite, and EDTA on bovine pulp and

dentin. Journal of Endodontics 2003; 29:334–7.

29.T. K. Machnick, M. Torabinejad, C. A. Munoz, S. Shabahang. Effect of MTAD

on flexural strength and modulus of elasticity of dentin. Journal of Endoddontics

2003; 29:747–50.

30.S. Shabahang, M. Pouresmail, M. Torabinejad. In vitro antimicrobial efficacy of

MTAD and sodium hypochlorite. Journal of Endodontics 2003; 29:450–2.

23

FIGURE LEGENDS:

Figure 1 (graph 1): Graphical representation of the fall in the percentage of

haemoglobin with the increase in the volume of the irrigant for all the three groups is

shown in Graph 1.

Documents

cytotoxicity