*Edited by Steve Kerrigan and Niamh Moran*

Platelets play a key role in thrombosis and haemostasis. However recent evidence clearly demonstrates that the functional role of platelets extends to many other processes in the body. With an internationally recognised list of contributing authors, The Non-Thrombotic Role of Platelets in Health and Disease, is a unique and definitive source of state-of-the-art knowledge about the additional role of platelets outside thrombosis and haemostasis. The intended audience for The Non-Thrombotic Role of Platelets in Health and Disease includes platelet biologists, microbiologists, immunologists, haematologists, oncologists, respiratory physicians, cardiologists, neurobiologists, tissue engineers, as well as students and fellows in these areas.

ISBN 978-953-51-2208-1

The Non-Thrombotic Role of Platelets in Health and Disease

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**The Importance of Final Irrigation with Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal**

Aleksandar Mitić1, Nadica Mitić1, Slavoljub Živković2, Jelena Milašin3, Jovanka Gašić1, Vladimir Mitić4, Tatjana Tanić4 and Jelena Popović1 *1Department of Restorative Dentistry and Endodontics, Clinic of Dentistry, Medical Faculty, University of Nis, Nis, 2Department of Restorative Dentistry and Endodontics, Faculty of Dentistry, University of Belgrade, Belgrade, 3Institute of Genetics, Faculty of Dentistry, Univesity of Belgrade, 4Department of Jaw Orthopaedics, Dental Hospital, Niš Serbia*

#### **1. Introduction**

284 Oral Health Care – Pediatric, Research, Epidemiology and Clinical Practices

Jonsson, T. and T. J. Sigurdsson (2004). Autotransplantation of premolars to premolar sites.

Josefsson, E., V. Brattstrom, et al. (1999). Treatment of lower second premolar agenesis by

Kallu, R., F. Vinckier, et al. (2005). Tooth transplantations: a descriptive retrospective study.

Kim, E., J. Y. Jung, et al. (2005). Evaluation of the prognosis and causes of failure in 182 cases

Magheri, P., R. Grandini, et al. (2001). Autogenous dental transplants: description of a

Marzola, C. (1968). [Dental reimplantation. Surgical, clinical and radiographic

Northway, W. (2002). Autogenic dental transplants*. Am J Orthod Dentofacial Orthop* 121(6):

Paulsen, H. U. (2001). Autotransplantation of teeth in orthodontic treatment. *Am J Orthod*

Paulsen, H. U., J. O. Andreasen, et al. (1995). Pulp and periodontal healing, root

Paulsen, H. U., X. Q. Shi, et al. (2001). Eruption pattern of autotransplanted premolars

Thomas, S., S. R. Turner, et al. (1998). Autotransplantation of teeth: is there a role*? Br J*

Tsukiboshi, M. (2002). Autotransplantation of teeth: requirements for predictable success.

development and root resorption subsequent to transplantation and orthodontic rotation: a long-term study of autotransplanted premolars. *Am J Orthod Dentofacial*

visualized by radiographic color-coding. *Am J Orthod Dentofacial Orthop* 119(4): 338-

clinical case*. Int J Periodontics Restorative Dent* 21(4): 367-371.

considerations*]. Rev Bras Odontol* 25(153): 254-269.

*Orthop* 125(6): 668-675.

*Endod* 100(1): 112-119.

*Orthop* 108(6): 630-640.

*Orthod* 25(4): 275-282.

*Int J Oral Maxillofac Surg* 34(7): 745-755.

*Dentofacial Orthop* 119(4): 336-337.

Tsukiboshi, M., Ed. (2001). *Autotransplantation of teeth*.

*Dent Traumatol* 18(4): 157-180.

57(2): 111-115.

592-593.

345.

A long-term follow-up study of 40 consecutive patients. *Am J Orthod Dentofacial*

autotransplantation: four-year evaluation of eighty patients. *Acta Odontol Scand*

of autogenous tooth transplantation. *Oral Surg Oral Med Oral Pathol Oral Radiol*

In primary tooth root canal infections, the largest number of microorganisms can be found in main root canal. However, a considerable portion of infection is located deeper, in the lateral canals, apical ramifications and dentinal tubules (Hülsmann et al., 1997; Matuse et al., 2003; Živković et al., 2005). It is precisely those anatomic variabilities and physiological specificities of endodontic and periodontal tissues that make impeding factors in endodontic infection resolving (Gašić et al., 2003; Mitić et al., 2009; Chacker 1974; De Deus, 1975). All chemomechanical techniques of canal preparation leave considerable amounts of debris and smear layer (Živković et al., 2005; Mitić, 2008).

Smear layer is a layer of debris remaining on dentin during instrumentation, and consists of dentin particles, remnants of vital or necrotic pulp tissues, bacteria and their components (Nešković & Živković, 2009; Abdullah et al., 2005; Calt & Serper, 2002; Fouad et al., 2002; Jacinto, 2003; Love, 2001; Portenier et al., 2001, 2003; Spratt et al., 2001; Shabahang et al., 2003.)

In clinical practice, instrumentation and irrigation of canal within endodontic treatment is time-consuming and the most demanding treatment phase (Mitić et al., 2011; Živković et al., 2005; Morazin et al., 1994; Baumgartner & Mader, 1987).

Smear layer is an ideal medium for growth and proliferation of microorganisms, and therefore should be removed before the final root canal obturation to reduce the microorganisms present in the root canal, to improve the adhesion of root canal sealers to the root canal walls and to reduce the apical and coronal microleakage (Hülsmann et al., 1997; Takeda et al., 1999; Mitić, 2010.).

The Importance of Final Irrigation with

• Score 1 - No smear layer, dentinal tubules open;

Fig. 1. Tooth roots notched with diamand discs

• Score 2 - Small amount of smear layer, several dentinal tubules open;

Disinfection of crowns and cavities was done by 3% natrium hypochlorite.

different magnifications.

dentin was scored as:

tubules open;

mean scores.

rubber dam.

p<0.05.

Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal 287

remove the superficial debris accumulated during cutting. Canals were dried with compressed air. Using the separation pliers, tooth roots were longitudinally grooved, into the mesial and distal halves. Each half of a sample was further fixed to a bed, coated with gold and viewed under the a scanning electron microscope JEOL-JSM-5300. The apex, middle and coronal thirds of all samples were analyzed; photomicrographs were taken at

According to the crictria specified by Hülsmann (1997), the smear layer of the root canal

• Score 3 - Homogenous smear layer covering the root canal wall, only few dentinal

For statistical analysis, Software SPSS 15.0 was used. Statistical significance was taken at

Diagnosis was established based on the anamnesis, objective examination and additional diagnostic methods (examination of vitality by electrotest and radiography). The patients were aged 29-56 years. All the examined teeth were diagnosed with primary apical periodontitis, with destructed tooth crowns but without fillings and prosthetic restorations. All interventions were performed maintaining a dry working field using a

• Score 4 - Dentinal wall completely covered by smear layer, no dentinal tubules open; • Score 5 - Heavy, non-homogenous smear layer completely covering the root canal wall. Statistical analysis included the comparison of the mean scores for the seven groups of analyzed samples; Kruskal Wallis nonparametric test was also used. Post hoc analysis was performed by Mann Whitney U test to determine single, intergroup differences among the

Medication aspect of chemomechanical root canal treatment involves the irrigation of root canal and removal of smear layer by the application of various preparations. The efficacy of irrigants is determined by numerous factors: concentration, pH value, root canal length, "age" of dentin tissue and time of application. One should bear in mind the fact that dentin tissue reduces the antimicrobial effect of various irrigants. Dentin hydroxyapatite possesses a buffering capacity, as it can donate protons, cause a pH change, and reduce the effects of various chemical agents when making contact with the dentinal wall (Haapasalo et al., 2000). The most frequently used irrigants with organolithic effects are sodium hypochlorite, hydrogen peroxide, chloramines, chlorhexidine. The final irrigants with mineralolithic effect are 17% NaEDTA, 10% citric acid, and the solution of recent date - MTAD (Biopure, Tulsa Dentsply, Tulsa OK, USA) – a combination of tetracyclines containing weak organic acids and anion-active substances (Torabinejad et al., 2002, 2003; Kando et al., 1991; Di Lenardda, 2000; Mitić et al., 2009; Yamaguchi et al., 1996; Haapasalo et al., 2005; Mitić, 2010).

The purpose of the present research was to analyze the surface of intracanal dentin after instrumentation and irrigation by organolithic agents (2.5% sodium hypochlorite, 3% hydrogen peroxide, 2% chlorhexidine) and final irrigation by mineralolithic effect solutions (17% NaEDTA, 10% citric acid and MTAD solution).

#### **2. Methods**

#### **2.1. Materials**

In the research, 145 freshly extracted single-rooted and double-rooted maxillary and mandibular human teeth were used. The teeth were extracted for orthodontic reasons in children of both sexes, aged 9-12 years.

The preparation of biomaterial involved storing of teeth in the sterile isotonic saline solution at 4C, without the use of fixatives. All samples were prepared by one operator. The preparation of root canal was carried out by hand K-files, sized # 15-40 (Display, Maillefer, Ballaigues, Switzerland) and rotary instrumentation. Root canals were instrumented using a standard step-back technique, while the apex third was enlarged up to # 30. For canal irrigation, we used special irrigation needles with lateral perforations. They ensured an immediate contact between solution and intracanal dentin even in the apical region, improving thus the debridement of the entire root canal wall.

Teeth were divided into two groups. A control group (n=40) was divided into four subgroups (a, b, c, d) for the purpose of quantitative assessment of smear layer on the samples after manual and rotary root canal instrumentation without irrigation (a, b) and after rinsing with sterile saline solution solution (c, d) (positive control).

The second, experimental group (n=105) was divided into seven subgroups (A,B,C,D,E,F,G), in which process the samples from the subgroups A,B,C were rinsed only with solutions with organolithic effect, while the samples from the subgroups D,E,F and G, besides irrigation with organolithic solutions (2.5% sodium hypochlorite, 3% hydrogen peroxide, 2% chlorhexidine – 2ml), were finally rinsed by mineralolithic effect solutions (17% NaEDTA, 10% citric acid and MTAD - 2ml). The samples rinsed with 5.25% NaOCl and 17% NaEDTA served as (negative control).

After chemomechanical root canal preparation, teeth crowns were removed with a diamond disk at the cement - enamel junction. All the samples were irrigated with distilled water to remove the superficial debris accumulated during cutting. Canals were dried with compressed air. Using the separation pliers, tooth roots were longitudinally grooved, into the mesial and distal halves. Each half of a sample was further fixed to a bed, coated with gold and viewed under the a scanning electron microscope JEOL-JSM-5300. The apex, middle and coronal thirds of all samples were analyzed; photomicrographs were taken at different magnifications.

According to the crictria specified by Hülsmann (1997), the smear layer of the root canal dentin was scored as:

• Score 1 - No smear layer, dentinal tubules open;

286 Oral Health Care – Pediatric, Research, Epidemiology and Clinical Practices

Medication aspect of chemomechanical root canal treatment involves the irrigation of root canal and removal of smear layer by the application of various preparations. The efficacy of irrigants is determined by numerous factors: concentration, pH value, root canal length, "age" of dentin tissue and time of application. One should bear in mind the fact that dentin tissue reduces the antimicrobial effect of various irrigants. Dentin hydroxyapatite possesses a buffering capacity, as it can donate protons, cause a pH change, and reduce the effects of various chemical agents when making contact with the dentinal wall (Haapasalo et al., 2000). The most frequently used irrigants with organolithic effects are sodium hypochlorite, hydrogen peroxide, chloramines, chlorhexidine. The final irrigants with mineralolithic effect are 17% NaEDTA, 10% citric acid, and the solution of recent date - MTAD (Biopure, Tulsa Dentsply, Tulsa OK, USA) – a combination of tetracyclines containing weak organic acids and anion-active substances (Torabinejad et al., 2002, 2003; Kando et al., 1991; Di Lenardda, 2000; Mitić et al., 2009; Yamaguchi et al., 1996; Haapasalo et al., 2005; Mitić,

The purpose of the present research was to analyze the surface of intracanal dentin after instrumentation and irrigation by organolithic agents (2.5% sodium hypochlorite, 3% hydrogen peroxide, 2% chlorhexidine) and final irrigation by mineralolithic effect solutions

In the research, 145 freshly extracted single-rooted and double-rooted maxillary and mandibular human teeth were used. The teeth were extracted for orthodontic reasons in

The preparation of biomaterial involved storing of teeth in the sterile isotonic saline solution at 4C, without the use of fixatives. All samples were prepared by one operator. The preparation of root canal was carried out by hand K-files, sized # 15-40 (Display, Maillefer, Ballaigues, Switzerland) and rotary instrumentation. Root canals were instrumented using a standard step-back technique, while the apex third was enlarged up to # 30. For canal irrigation, we used special irrigation needles with lateral perforations. They ensured an immediate contact between solution and intracanal dentin even in the apical region,

Teeth were divided into two groups. A control group (n=40) was divided into four subgroups (a, b, c, d) for the purpose of quantitative assessment of smear layer on the samples after manual and rotary root canal instrumentation without irrigation (a, b) and after rinsing

The second, experimental group (n=105) was divided into seven subgroups (A,B,C,D,E,F,G), in which process the samples from the subgroups A,B,C were rinsed only with solutions with organolithic effect, while the samples from the subgroups D,E,F and G, besides irrigation with organolithic solutions (2.5% sodium hypochlorite, 3% hydrogen peroxide, 2% chlorhexidine – 2ml), were finally rinsed by mineralolithic effect solutions (17% NaEDTA, 10% citric acid and MTAD - 2ml). The samples rinsed with 5.25% NaOCl and 17% NaEDTA

After chemomechanical root canal preparation, teeth crowns were removed with a diamond disk at the cement - enamel junction. All the samples were irrigated with distilled water to

(17% NaEDTA, 10% citric acid and MTAD solution).

improving thus the debridement of the entire root canal wall.

with sterile saline solution solution (c, d) (positive control).

children of both sexes, aged 9-12 years.

served as (negative control).

2010).

**2. Methods 2.1. Materials**

Statistical analysis included the comparison of the mean scores for the seven groups of analyzed samples; Kruskal Wallis nonparametric test was also used. Post hoc analysis was performed by Mann Whitney U test to determine single, intergroup differences among the mean scores.

For statistical analysis, Software SPSS 15.0 was used. Statistical significance was taken at p<0.05.

Diagnosis was established based on the anamnesis, objective examination and additional diagnostic methods (examination of vitality by electrotest and radiography). The patients were aged 29-56 years. All the examined teeth were diagnosed with primary apical periodontitis, with destructed tooth crowns but without fillings and prosthetic restorations. All interventions were performed maintaining a dry working field using a rubber dam.

Disinfection of crowns and cavities was done by 3% natrium hypochlorite.

Fig. 1. Tooth roots notched with diamand discs

\*Without irrigation

Table 1. Experimental protocol for control group samples

The Importance of Final Irrigation with

Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal 289

Table 2. Experimental protocol for group II (experimental) samples

\*Without irrigation

Table 1. Experimental protocol for control group samples

Table 2. Experimental protocol for group II (experimental) samples

The Importance of Final Irrigation with

irrigation (p<0.001).

Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal 291

Statistical analysis showed that the experimental group treated with MTAD as the final irrigation had significantly cleaner walls compared to control group samples (p<0.001). The analysis of results showed that there was statistically significant difference in the mean scores among the examined groups of samples (χ2=50.674; p<0.001). The lowest score, and

Mann Whitney **U** test determined statistically significant differences in scores among the groups, and the best results were obtained in the teeth in which MTAD was used as the final

Group/subgroup N Chemomechanical treatment 5 4 3 2 1

I/A 10 Manual treatment without irrigation 8 2 0 0 0

I/B 10 Engine driven treatment without irrigation 9 1 0 0 0

I/C 10 Manual treatment + Saline solution 3 7 0 0 0

I/D 10 Engine driven treatment + Saline solution 4 4 2 0 0

Table 3. Values of quantitative estimation of smear layer and dentin debris for group I

N *X* SD 95%C.I. Min Max Sig.

I/A 10 4.80 0.42 4.50-5.10 4 5 A

I/B 10 4.90 0.32 4.67-5.13 4 5 B, C

I/C 10 4.30 0.48 3.95-4.65 4 5 B

I/D 10 4.20 0.79 3.64-4.76 3 5 A, C

Table 4. Comparison of values of quantitative estimation of smear layer and dentin debris in

By comparing the quantitative estimation values obtained for smear layer and dentin debris in the group I (control) samples – subgroups A, B, C and D using the ANOVA test, a statistically significant difference was found in the mean values among the groups I/A and

**3.1.1 Ultrastructural presentation of intracanal dentin surface after manual treatment**

After tooth root canal treatment using the hand driven instruments, canal walls were covered with large particles of dentin debris and smear layer of irregular surface found at all levels of intracanal dentin. The size of dentin debris particles was 2-6 μm. Dentinal tubule

(control group) samples – subgroups A, B ,C and D

A (I/A vs I/D); B (I/B vs I/C); C (I/B vs I/D);

I/D, I/B and I/C, I/B and I/D.

**without irrigation**

group I (control samples - subgroups A,B,C and D: ANOVA test

**3.1. Ultrastructural presentation of intracanal dentin**

therewith the most favorable outcome, was found in the group F (Tab. 2).

Fig. 2. Sample prepared for evaporation

Fig. 3. Placing samples on appropriate supporter

#### **3. Result**

The results obtained in this study are presented in tables 1-7 and figures 1-15. In the first group of control samples subgroups (A, B, C, D), it was observed that root canal walls were covered with substantial amounts of dentin debris, where smear layer completely closed the openings of dentinal tubules. Such presentation of dentin surface is described in literature as "bark tree". Eight samples were scored 5, while two samples were scored 4.

In the experimental group, the poorest results were obtained after the irrigation of walls with organolithic effect solutions. The most favorable outcome of the procedure was observed in the group where canal irrigation during instrumentation was done with organolithic effect solutions, and final irrigation with mineralolithic effect irrigating agents, in which process the most optimal combination of irrigation solutions was 3% H2O2 + 2% CHX + MTAD (1.10±0.31), which is a good choice of irrigants in endodontic clinical practice.

The results obtained in this study are presented in tables 1-7 and figures 1-15. In the first group of control samples subgroups (A, B, C, D), it was observed that root canal walls were covered with substantial amounts of dentin debris, where smear layer completely closed the openings of dentinal tubules. Such presentation of dentin surface is described in literature as

In the experimental group, the poorest results were obtained after the irrigation of walls with organolithic effect solutions. The most favorable outcome of the procedure was observed in the group where canal irrigation during instrumentation was done with organolithic effect solutions, and final irrigation with mineralolithic effect irrigating agents, in which process the most optimal combination of irrigation solutions was 3% H2O2 + 2% CHX + MTAD (1.10±0.31), which is a good choice of irrigants in endodontic

"bark tree". Eight samples were scored 5, while two samples were scored 4.

Fig. 2. Sample prepared for evaporation

Fig. 3. Placing samples on appropriate supporter

**3. Result**

clinical practice.

Statistical analysis showed that the experimental group treated with MTAD as the final irrigation had significantly cleaner walls compared to control group samples (p<0.001).

The analysis of results showed that there was statistically significant difference in the mean scores among the examined groups of samples (χ2=50.674; p<0.001). The lowest score, and therewith the most favorable outcome, was found in the group F (Tab. 2).

Mann Whitney **U** test determined statistically significant differences in scores among the groups, and the best results were obtained in the teeth in which MTAD was used as the final irrigation (p<0.001).

Table 3. Values of quantitative estimation of smear layer and dentin debris for group I (control group) samples – subgroups A, B ,C and D

A (I/A vs I/D); B (I/B vs I/C); C (I/B vs I/D);

Table 4. Comparison of values of quantitative estimation of smear layer and dentin debris in group I (control samples - subgroups A,B,C and D: ANOVA test

By comparing the quantitative estimation values obtained for smear layer and dentin debris in the group I (control) samples – subgroups A, B, C and D using the ANOVA test, a statistically significant difference was found in the mean values among the groups I/A and I/D, I/B and I/C, I/B and I/D.

#### **3.1. Ultrastructural presentation of intracanal dentin**

#### **3.1.1 Ultrastructural presentation of intracanal dentin surface after manual treatment without irrigation**

After tooth root canal treatment using the hand driven instruments, canal walls were covered with large particles of dentin debris and smear layer of irregular surface found at all levels of intracanal dentin. The size of dentin debris particles was 2-6 μm. Dentinal tubule

The Importance of Final Irrigation with

**treatment and irrigation with saline solution**

**3% H2O2 and final irrigation with 2,5% NaOCl**

smear layer can be seen.

Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal 293

Fig. 6. SEM micrographs of coronal (K), middle (S) and apical (A) regions of canal walls after manual treatment (using K files) and irrigation with saline solution. A smaller quantity of

**3.1.4 Ultrastructural presentation of intracanal dentin surfaces after engine driven**

Fig. 7. SEM micrographs of coronal (K), middle (S) and apical (A) thirds of canal walls following engine driven treatment and irrigation with saline solution. Small particles (K) of dentinal debris, as well as larger ones (S, A), and abundant smear layer can be observed.

**3.1.5 Ultrastructural presentation of intracanal dentin surfaces after the irrigation with**

After manual treatment and irrigation during instrumentation using 3% H2O2 and final irrigation with 2,5% NaOCl, it can be noted that larger amount of debris in the coronal

Fig. 8. SEM micrograps of coronal (K), middle (S) and apical (A) thirds of canal walls following the treatment and irrigation with 3% H2O2 and final irrigation with 2,5% NaOCl

Dentinal tubule openings of irregular diameters and infundibular shape

After intracanal engine driven treatment using NiTi instruments and irrigation during instrumentation with saline solution, smaller particles of dentinal debris and abundant

superficial debris and inhomogeneous smear layer distributed in all root regions

openings were not visible; dentin debris with a "tree bark" configuration was observed at the level of the apical third.

Fig. 4. SEM micrographs of coronal (K), middle (S) and apical (A) thirds of canal walls after manual treatment using K files without irrigation. Massive accumulation of dentin debris with large particles, with the underlying smear layer. Dentinal tubules invisible

#### **3.1.2 Ultrastructural presentation of intracanal dentin surfaces after engine driven treatment without irrigation**

Intracanal engine driven treatment of tooth root canal using NiTi instruments, without irrigation, leaves massive accumulation of dentin debris, with the underlying smear layer of thicker, more compact structure having large dentin particles distributed throughout the root canal. Dentinal tubules are completely closed. In the apical regions, there is a large amount of dentinal debris in the form of "plug".

Fig. 5. SEM micrographs of coronal (K), middle (S) and apical thirds(A) of the canal walls after engine driven treatment using NiTi files without irrigation. Massive accumulation of dentinal debris

#### **3.1.3 Ultrastructural presentation of intracanal dentinal surfaces following manual treatment using saline solution**

After manual root canal treatment with K files and irrigation with saline solution during instrumentation, a smaller amount of superficial debris can be noted as well as the presence of inhomogeneous smear layer in all root regions. Only a few dentinal canal openings can be seen, completelly or partially covered with smear layer and debris (coronal and middle root regions), while the apical region contains larger amounts of surface debris and smear layer.

openings were not visible; dentin debris with a "tree bark" configuration was observed at

Fig. 4. SEM micrographs of coronal (K), middle (S) and apical (A) thirds of canal walls after manual treatment using K files without irrigation. Massive accumulation of dentin debris

**3.1.2 Ultrastructural presentation of intracanal dentin surfaces after engine driven**

Fig. 5. SEM micrographs of coronal (K), middle (S) and apical thirds(A) of the canal walls after engine driven treatment using NiTi files without irrigation. Massive accumulation of

**3.1.3 Ultrastructural presentation of intracanal dentinal surfaces following manual**

After manual root canal treatment with K files and irrigation with saline solution during instrumentation, a smaller amount of superficial debris can be noted as well as the presence of inhomogeneous smear layer in all root regions. Only a few dentinal canal openings can be seen, completelly or partially covered with smear layer and debris (coronal and middle root regions), while the apical region contains larger amounts of surface debris and smear layer.

Intracanal engine driven treatment of tooth root canal using NiTi instruments, without irrigation, leaves massive accumulation of dentin debris, with the underlying smear layer of thicker, more compact structure having large dentin particles distributed throughout the root canal. Dentinal tubules are completely closed. In the apical regions, there is a large

with large particles, with the underlying smear layer. Dentinal tubules invisible

the level of the apical third.

**treatment without irrigation**

dentinal debris

**treatment using saline solution**

amount of dentinal debris in the form of "plug".

Fig. 6. SEM micrographs of coronal (K), middle (S) and apical (A) regions of canal walls after manual treatment (using K files) and irrigation with saline solution. A smaller quantity of superficial debris and inhomogeneous smear layer distributed in all root regions

#### **3.1.4 Ultrastructural presentation of intracanal dentin surfaces after engine driven treatment and irrigation with saline solution**

After intracanal engine driven treatment using NiTi instruments and irrigation during instrumentation with saline solution, smaller particles of dentinal debris and abundant smear layer can be seen.

Fig. 7. SEM micrographs of coronal (K), middle (S) and apical (A) thirds of canal walls following engine driven treatment and irrigation with saline solution. Small particles (K) of dentinal debris, as well as larger ones (S, A), and abundant smear layer can be observed. Dentinal tubule openings of irregular diameters and infundibular shape

#### **3.1.5 Ultrastructural presentation of intracanal dentin surfaces after the irrigation with 3% H2O2 and final irrigation with 2,5% NaOCl**

After manual treatment and irrigation during instrumentation using 3% H2O2 and final irrigation with 2,5% NaOCl, it can be noted that larger amount of debris in the coronal

Fig. 8. SEM micrograps of coronal (K), middle (S) and apical (A) thirds of canal walls following the treatment and irrigation with 3% H2O2 and final irrigation with 2,5% NaOCl

The Importance of Final Irrigation with

**irrigation with 2,5% NaOCl + 10% citric acid**

dentin, formations of calcium citrate are produced

structure

Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal 295

Fig. 10. SEM micrographs of canal walls after treatment and irrigation with 2,5% NaOCl and final irrigation using17% NaEDTA solution. Dentinal debris and smear layer comletely removed. Dentinal tubules clearly open, of regular shape; preserved, smooth dentinal

**3.1.8 Ultrastructural presentation of intracanal dentin surfaces after treatment and**

Fig. 11. SEM micrographs of intracanal dentine after irrigation with 2,5% NaOCl and 10% citric acid in duration of 60 sec. Small dentin particles present with the underlying smear layer. Dentinal tubules open, of eneven diameter and shape. Because of chelating Ca+ from

Citric acid (10%) is efficient in removing the smear layer from the root canal walls and complete cleansing of the canal system, and, therefore, can be used as a final irrigant during endodontic treatment. In order to avoid agressive etching and potential erosion of dentin, the time of citric acid action must be limited from 20 seconds to 1 minute. The basic problem when applying the citric acid in the intracanal irrigation is the acidity of solution and a possibility of accidental contact with the mouth cavity soft issues. Combined application with NaOCl can bring about sudden neutralization, pH changes and releasing the chloride

region was removed; however, the smear layer is present. The open dentinal tubules show irregular shape and diameter. In the middle and apical thirds of the coronal region considerable amounts of surface debris attached to smear layer can be observed, which largely blocks the openings of dentinal tubules. There is a lack of mineralolithic effect, which results in impure intracanal dentin surface.

#### **3.1.6 Chlorhexidine (CHX) as intracanal irrigants**

Chlorhexidine is gluconate salt, and as an intracanal irrigants it is used in the form of bisbiguanide. This biocide has prolonged antibacterial efficacy at pH - 5.5-7.0. Ultrastructural presentation of intracanal dentinal surfaces after the irrigation with 3% H2O2 and final irrigation with 2% CHX.

After the manual root canal treatment and irrigation during instrumentation using 3% H2O2 and final irrigation with 2% CHX, the largest part of debris was removed. However, small particles of the smear layer, sized 1-2 µm, can be noted in all the samples, which results in not so smooth surface of the intertubular dentin. Dentinal tubule openings are not clearly limited, and inside the tubules some small particles of precipitate can be seen, produced during the reaction between hydrogen peroxide and chlorhexidine.

Having observed the dentinal wall from the coronal to the apical third, it was found that the amount of the smear layer was increased from the middle towards the apical third. In the middle third, the openings of dentinal tubules are of uneven diameter and irregular shape. The largest quantity of the smear layer in the form of "plug " is in the apical thid, wherein the created precipitate is incorporated into the dentinal tubules. In the apical third, the openings of dentinal tubules cannot be seen.

Fig. 9. SEM micrographs of the coronal (K), middle (S) and apical (A) thirds of the canal wall after the treatment and irrigation with 3% H2O2 and final irrigation with 2% CHX solution. Only small particles of dentinal debris can be partly seen as well as the smear layer on intertubular dentin; precipitates present inside the dentinal tubules

#### **3.1.7 Ultrastructural presentation of intracanal dentin surfaces after treatment, irrigation with 2,5% NaOCl and final irrigation using 17%Na EDTA**

Intracanal dentin surfaces in all regions, after the treatment and irrigation with 2,5% NaOCl and final irrigation with 17% NaEDTA solution, show preserved and clean structural dentin surface, open dentinal tubules of regular and even lumen.

region was removed; however, the smear layer is present. The open dentinal tubules show irregular shape and diameter. In the middle and apical thirds of the coronal region considerable amounts of surface debris attached to smear layer can be observed, which largely blocks the openings of dentinal tubules. There is a lack of mineralolithic effect, which

Chlorhexidine is gluconate salt, and as an intracanal irrigants it is used in the form of bisbiguanide. This biocide has prolonged antibacterial efficacy at pH - 5.5-7.0. Ultrastructural presentation of intracanal dentinal surfaces after the irrigation with 3% H2O2 and final

After the manual root canal treatment and irrigation during instrumentation using 3% H2O2 and final irrigation with 2% CHX, the largest part of debris was removed. However, small particles of the smear layer, sized 1-2 µm, can be noted in all the samples, which results in not so smooth surface of the intertubular dentin. Dentinal tubule openings are not clearly limited, and inside the tubules some small particles of precipitate can be seen, produced during the reaction between hydrogen peroxide and

Having observed the dentinal wall from the coronal to the apical third, it was found that the amount of the smear layer was increased from the middle towards the apical third. In the middle third, the openings of dentinal tubules are of uneven diameter and irregular shape. The largest quantity of the smear layer in the form of "plug " is in the apical thid, wherein the created precipitate is incorporated into the dentinal tubules. In the apical third, the

Fig. 9. SEM micrographs of the coronal (K), middle (S) and apical (A) thirds of the canal wall after the treatment and irrigation with 3% H2O2 and final irrigation with 2% CHX solution. Only small particles of dentinal debris can be partly seen as well as the smear layer on

Intracanal dentin surfaces in all regions, after the treatment and irrigation with 2,5% NaOCl and final irrigation with 17% NaEDTA solution, show preserved and clean structural dentin

**3.1.7 Ultrastructural presentation of intracanal dentin surfaces after treatment,**

intertubular dentin; precipitates present inside the dentinal tubules

**irrigation with 2,5% NaOCl and final irrigation using 17%Na EDTA**

surface, open dentinal tubules of regular and even lumen.

results in impure intracanal dentin surface.

openings of dentinal tubules cannot be seen.

irrigation with 2% CHX.

chlorhexidine.

**3.1.6 Chlorhexidine (CHX) as intracanal irrigants**

Fig. 10. SEM micrographs of canal walls after treatment and irrigation with 2,5% NaOCl and final irrigation using17% NaEDTA solution. Dentinal debris and smear layer comletely removed. Dentinal tubules clearly open, of regular shape; preserved, smooth dentinal structure

#### **3.1.8 Ultrastructural presentation of intracanal dentin surfaces after treatment and irrigation with 2,5% NaOCl + 10% citric acid**

Citric acid (10%) is efficient in removing the smear layer from the root canal walls and complete cleansing of the canal system, and, therefore, can be used as a final irrigant during endodontic treatment. In order to avoid agressive etching and potential erosion of dentin, the time of citric acid action must be limited from 20 seconds to 1 minute. The basic problem when applying the citric acid in the intracanal irrigation is the acidity of solution and a possibility of accidental contact with the mouth cavity soft issues. Combined application with NaOCl can bring about sudden neutralization, pH changes and releasing the chloride

Fig. 11. SEM micrographs of intracanal dentine after irrigation with 2,5% NaOCl and 10% citric acid in duration of 60 sec. Small dentin particles present with the underlying smear layer. Dentinal tubules open, of eneven diameter and shape. Because of chelating Ca+ from dentin, formations of calcium citrate are produced

The Importance of Final Irrigation with

Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal 297

Fig. 14. SEM presentation of apical third of intracanal dentin after manual treatment and

Table 5. Values of quantitative estimation of smear layer and dentinal debris for the

Group/subgroups N *X* SD 95%C.I. Min Max Sig. II/A (3%H2O2+2,5%NaOCl) 15 2.66 0.89 2.16-3.16 1.00 4.00 A,B,C,D,E,F II/B (3%H2O2 +2%CHX) 15 2.73 0.88 2.24-3.22 1.00 4.00 A,G,H,I,J,K II/C (2,5%NaOCl+17%NaEDTA) 15 1.20 0.41 0.97-1.42 1.00 2.00 A,G,L

II/D (2,5%NaOCl) 15 3.80 0.86 3.32-4.27 3.00 5.00 C,H,L,M,N,O

Table 6. Comparison of quantitative estimation values of smear layer and dentinal debris on

By comparison of the quantitative estimation values of smear layer and dentinal debris on the samples of group II (experimental) – subgroups A, B, C, D, E, F and G, using the ANOVA test, it was found that the values obtained in the group II/A are statistically

the samples of group II (experimental) – subgroups A, B, C, D, E, F, and G: ANOVA

II/E (2,5%NaOCl +10% citric acid 15 1.26 0.45 1.01-1.52 1.00 2.00 D,I,M II/F (3%H2O2+2%CHX +MTAD) 15 1.06 0.25 0.92-1.20 1.00 2.00 E,J,N II/G (3%H2O2+MTAD) 15 1.00 0.00 1.00-1.00 1.00 1.00 F,K,O A (II/A vs II/B); B (II/A vs II/C); C (II/A vs II/D); D (II/A vs II/E); E (II/A vs II/F); F (II/A vs II/G);G (II/B vs II/C); H (II/B vs II/D); I (II/B vs II/E); J (II/B vs II/F); K (II/B vs II/G);L (II/C vs

samples of group II (experimental) – subgroups A, B, C, D, E, F and G

II/D); M (II/D vs II/E); N (II/D vs II/F); O (II/D vs II/G)

Group/subgroup N Irrigants 5 4 3 2 1 II/A 15 3%H2O2+2,5% NaOCl 0 2 3 8 2 II/B 15 3%H2O22% +CHX 0 1 3 9 2 II/C 15 2,5%NaOCl+17%NaEDTA 0 0 0 2 13 II/D 15 2,5%NaOCl 4 4 7 0 0 II/E 15 2,5%NaOCl +10% citric acid 0 0 0 4 11 II/F 15 3%H2O +2%CHX +MTAD 0 0 0 1 14 I/G 15 3%H2O2 + MTAD 0 0 0 0 15

irrigation with 2% CHX + 3% H2O2 and final irrigation with MTAD solution

gases. Higher concentration of citric acid can chelate Ca2+ from dentin and cause the formation of calcium citrate crystals in the root canal. Industrial products are: 19% citric acid solution – canal Clean (Ognapharma, Italy); 10% citric acid solution – Citric acid solution (Ultradent) for canal application, Cetrimide.

#### **3.1.9 Ultrastctural presentation of intracanal dentin surface after the irrigation with 3% H2O2 i 2% CHX and final irrigation with MTAD solution**

After the canal system instrumentation and irrigation with 3% H2O2 i 2% CHX and final irrigation with MTAD solution in duration of 1 minute, the results obtained are ideal in the coronal, middle and apical thirds of the tooth root canal. Dentinal debris and smear layer compley removed. Dentinal tubules open, of regular shape and even diameter (coronary third 3,5 µm, middle third 2,5-3 µm, apical third 2-2,5 µm). Dentinal structure preserved.

Fig. 12. SEM presentation of coronal third of intracanal dentin after manual treatment and irrigation with 2% CHX + 3% H2O2 and final irrigation with MTAD solution

Fig. 13. SEM presentation of middle third of intracanal dentin after manual treatment and irrigation with 2% CHX + 3% H2O2 and final irrigation with MTAD solution

gases. Higher concentration of citric acid can chelate Ca2+ from dentin and cause the formation of calcium citrate crystals in the root canal. Industrial products are: 19% citric acid solution – canal Clean (Ognapharma, Italy); 10% citric acid solution – Citric acid solution

**3.1.9 Ultrastctural presentation of intracanal dentin surface after the irrigation with 3%**

After the canal system instrumentation and irrigation with 3% H2O2 i 2% CHX and final irrigation with MTAD solution in duration of 1 minute, the results obtained are ideal in the coronal, middle and apical thirds of the tooth root canal. Dentinal debris and smear layer compley removed. Dentinal tubules open, of regular shape and even diameter (coronary third 3,5 µm, middle third 2,5-3 µm, apical third 2-2,5 µm). Dentinal structure

Fig. 12. SEM presentation of coronal third of intracanal dentin after manual treatment and

Fig. 13. SEM presentation of middle third of intracanal dentin after manual treatment and

irrigation with 2% CHX + 3% H2O2 and final irrigation with MTAD solution

irrigation with 2% CHX + 3% H2O2 and final irrigation with MTAD solution

(Ultradent) for canal application, Cetrimide.

preserved.

**H2O2 i 2% CHX and final irrigation with MTAD solution**

Fig. 14. SEM presentation of apical third of intracanal dentin after manual treatment and irrigation with 2% CHX + 3% H2O2 and final irrigation with MTAD solution

Table 5. Values of quantitative estimation of smear layer and dentinal debris for the samples of group II (experimental) – subgroups A, B, C, D, E, F and G

A (II/A vs II/B); B (II/A vs II/C); C (II/A vs II/D); D (II/A vs II/E); E (II/A vs II/F); F (II/A vs II/G);G (II/B vs II/C); H (II/B vs II/D); I (II/B vs II/E); J (II/B vs II/F); K (II/B vs II/G);L (II/C vs II/D); M (II/D vs II/E); N (II/D vs II/F); O (II/D vs II/G)

Table 6. Comparison of quantitative estimation values of smear layer and dentinal debris on the samples of group II (experimental) – subgroups A, B, C, D, E, F, and G: ANOVA

By comparison of the quantitative estimation values of smear layer and dentinal debris on the samples of group II (experimental) – subgroups A, B, C, D, E, F and G, using the ANOVA test, it was found that the values obtained in the group II/A are statistically

The Importance of Final Irrigation with

the treatment of infected root canals.

**5. Clinical recommendations**

parial evacuation of debris.

complete removal of the smear layer.

layer.

**4. Conclusion**

Mineralolithic Effect Agents During Chemomechanical Treatment of Tooth Root Canal 299

infected canal system (*Porphyromonas gingivalis, Acgregatibacter actinomycetemcomitans, Tanerella forsythensis, Prevotella intermedia, Treponema denticola* and *Enterococcus faecalis)* before and after the irrigation with MTAD solution. Analysis of results and estimation of antibacterial efficacy of MTAD solution gives the clinical reference to this final irrigant in

By comparing the frequency of occurrence of certain bacteria in root canals, before and after the irrigation with MTAD solution, statistically significant decrease of *Prevotella intermedia* (36% vs 0%),*Tanerella forsythenis* (24% vs 0%) and *Enterococcus faecalis* (60% vs 20%) was found. The presence of other bacteria was also decreased, but not statistically significant.

Based on the results obtained by SEM and statistical data processing, it can be concluded that the final irrigation of root canal system with mineralolithic effect irrigations must be a mandatory part of endodontic protocol. The best results and outstanding efficacy were demonstrated with MTAD solution. In combination with CHX and H2O2, it completely removes the smear layer from root canal walls, where the dentin surface structure remains preserved and openings of dentinal tubules are of even diameters and regular shapes. All mineralolithic solutions for final irrigation are used in duration of one minute, as longer exposure of dentin to these agents can bring about unwanted erosive changes and compromise the entire endodontic procedure. By regular use of final irrigating agents, complete efficacy in removal of smear layer from root canal system could be achieved.

• When performing the manual and endgine driven instrumentation of the root canal, dentinal debris and smear layer are produced at all the levels of the intraradix region;

• Irrigation by using organolithic agents alone cannot completely remove the smear

• The combination of organolithic with mineralolithic agents has shown as the most efficient in the removal of smear layer at all the levels of the intraradix region. • The combined application of hydrogene peroxide (and clorehexidine) during instrumentation and final irrigation with MTAD solution in duration of 1 minute results in

• MTAD solution as the final irrigant meets all the standards for good irrigant proscribed by the endodontic protocol, which means that it preserves the structure of dentine,

• MTAD solution, used as the final intracanal antiseptic in duration of 1 minute, efficiently removes the smear layer, in the case of which the intracanal structure

removes the smear layer and possesses the satisfactory antimicrobial properties. • After the chemomechanic treatment and irrigation of the root canal using the MTAD solution, statistically significant decrease of *Enterococcus faecalis, Prevotella intermedia*  and *Tanerella forsythenis* was found, while in cases of *Treponema denticola, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis* the antibacterial efficacy of MTAD

solution was considerable but not statistically significant.

they are not different in respect to the amount but presentation and structure. • Saline solution applied as an irrigant exerts only the mechanical effect of removal and

significantly different from the results obtained in all other groups. Mean value obtained in the group II/A was statistically significantly lower compared to the values in the groups II/B and II/D. The values obtained in the group II/B are statistically significantly higher compared to the values obtained in the groups II/C, II/E, II/F and II/G, but lower than the values obtained in the group II/D. The values of group II/d are statistically significantly higher than the values obtained in all other groups. The values of groups II/F and II/G are lower compared to the values obtained in the groups II/A, II/B and II/D.

#### **3.2 Analysis of antimicrobial effect with MTAD in infected canal system using PCR technique**

With the aim to determine the antibacterial efficacy of MTAD solution using the PCR method, several most common endopathogenic microorganisms were identified in the

Table 7. Prevalence of microorganisms in infected root canals prior and after MTAD therapy

Fig. 15. Prevalence of microorganisms in infected root canals before and after therapy with MTAD

infected canal system (*Porphyromonas gingivalis, Acgregatibacter actinomycetemcomitans, Tanerella forsythensis, Prevotella intermedia, Treponema denticola* and *Enterococcus faecalis)* before and after the irrigation with MTAD solution. Analysis of results and estimation of antibacterial efficacy of MTAD solution gives the clinical reference to this final irrigant in the treatment of infected root canals.

By comparing the frequency of occurrence of certain bacteria in root canals, before and after the irrigation with MTAD solution, statistically significant decrease of *Prevotella intermedia* (36% vs 0%),*Tanerella forsythenis* (24% vs 0%) and *Enterococcus faecalis* (60% vs 20%) was found. The presence of other bacteria was also decreased, but not statistically significant.

### **4. Conclusion**

298 Oral Health Care – Pediatric, Research, Epidemiology and Clinical Practices

significantly different from the results obtained in all other groups. Mean value obtained in the group II/A was statistically significantly lower compared to the values in the groups II/B and II/D. The values obtained in the group II/B are statistically significantly higher compared to the values obtained in the groups II/C, II/E, II/F and II/G, but lower than the values obtained in the group II/D. The values of group II/d are statistically significantly higher than the values obtained in all other groups. The values of groups II/F and II/G are

**3.2 Analysis of antimicrobial effect with MTAD in infected canal system using PCR**

With the aim to determine the antibacterial efficacy of MTAD solution using the PCR method, several most common endopathogenic microorganisms were identified in the

*Agregatibacter* 8 32 4 16 1.72 0.185 *Prevotella intermedia* 9 36 0 0 0.002 *Porphyromonas gingivalis* 4 16 0 0 0.11 *Tanerella forsythenis* 6 24 0 0 0.022 *Enterococcus faecalis* 15 60 5 20 8.17 0.004 *Treponema denticola* 7 28 3 12 1.96 0.161 Table 7. Prevalence of microorganisms in infected root canals prior and after MTAD therapy

**Before After**

Fig. 15. Prevalence of microorganisms in infected root canals before and after therapy with

*Actinobacillus P. intermedia P. gingivalis T. forsythenis E. faecalis T. denticola*

**\***

Number of infected root canals

**N % N %**

**2** Before therapy After therapy p

**\*\***

χ

lower compared to the values obtained in the groups II/A, II/B and II/D.

**technique**

MTAD

**0**

**\*** - p < 0,05; **\*\*** - p < 0,01

**\*\***

**5**

**10**

**15**

**20**

Bacterial species

Based on the results obtained by SEM and statistical data processing, it can be concluded that the final irrigation of root canal system with mineralolithic effect irrigations must be a mandatory part of endodontic protocol. The best results and outstanding efficacy were demonstrated with MTAD solution. In combination with CHX and H2O2, it completely removes the smear layer from root canal walls, where the dentin surface structure remains preserved and openings of dentinal tubules are of even diameters and regular shapes. All mineralolithic solutions for final irrigation are used in duration of one minute, as longer exposure of dentin to these agents can bring about unwanted erosive changes and compromise the entire endodontic procedure. By regular use of final irrigating agents, complete efficacy in removal of smear layer from root canal system could be achieved.

#### **5. Clinical recommendations**

The Importance of Final Irrigation with

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## *Edited by Mandeep Singh Virdi*

Oral health care in pediatric dentistry deals with complete oral health, including preventive aspects for children right from their conception to adolescence, encompassing all the spheres of dentistry including various specialties. It also includes planning a preventive program at individual and community levels. The current research interests in oral health care include studies regarding the role of stem cells, tissue culture, and other ground-breaking technologies available to the scientific community in addition to traditional fields such as anatomy, physiology, and pharmaceuticals etc of the oral cavity. Public health and epidemiology in oral health care is about the monitoring of the general oral health of a community, general afflictions they are suffering from, and an overall approach for care and correction of the same. The oral health care-giver undertakes evaluation of conditions affecting individuals for infections, developmental anomalies, habits, etc. and provides corrective action in clinical conditions. The present work is a compendium of articles by internationally renowned and reputed specialists about the current developments in various fields of oral health care.

Oral Health Care - Pediatric, Research, Epidemiology and Clinical Practices

Oral Health Care

Pediatric, Research,

Epidemiology and Clinical Practices

*Edited by Mandeep Singh Virdi*

Photo by Bet\_Noire / iStock


**1. Introduction**

The socket is the mechanical interface between prosthesis and residual limb. It accounts for stability, ensures control over the prosthetic device, and determines its level of comfort. Thus, stump–socket interaction strongly influences the well-being and mobility of amputees [1]. To ensure the quality of socket fit, quantitative measures are advantageous. Pistoning or relative movement between residual lower limb and prosthesis is considered to be one indicative parameter of socket fit quality [2].

The most common techniques that are used to acquire data on the relative movement between residual limb surface and prosthetic socket are: motion capture [3–6], other optical means [7,8], inductive sensors [9–12], and vacuum pressure fluctuations of elevated vacuum suspension systems [11,13]. These approaches are subject to different drawbacks. For instance, vacuum pressure fluctuations cannot be used to evaluate the relative motion at specific or problematic locations within the interface. Additionally, despite the number of different measurement approaches, studies presenting gait cycle dependent data of relative motion during ambulation are scarce [3,4,9,14].

A concept for measuring the relative motion between residual limb surface and prosthetic socket at specific locations in dynamic gait situations is presented in [15]. The proposed concept is based on optical 2D-motion sensor units, whose applicability has been tested experimentally using a functional model consisting of one sensor unit on a test rig as well as in biomechanical substitute studies [16].

This paper presents the subsequently realized low-cost measurement system capable of interfacing with an array of sensors. The implementation of the optimized measurement chain is presented in Section 2. In Section 3, the measurement system is evaluated experimentally on a test rig regarding accuracy, precision, achievable sampling frequency, and overall reliability. A pilot study using seven sensors to measure the relative motion between residual limb and prosthetic socket is reported in Section 4. Based on a discussion of the results, the paper is concluded in Section 5 where an outlook to future work is also given.