SEM EVALUATION OF EFFECT OF 37% PHOSPHORIC ACID GEL, … · EDTA gel for 60 seconds. IIIa - 5 specimens to be etched with 10% maleic acid gel for 15 seconds. IIIb - 5 specimens to

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INTERNATIONAL DENTAL JOURNAL OF STUDENT’S RESEARCH| June‐Sep 2012| Volume 1| Issue 2

ORIGINAL ARTICLE

SEM EVALUATION OF EFFECT OF 37% PHOSPHORIC ACID GEL, 24% EDTA GEL AND 10% MALEIC ACID GEL ON THE ENAMEL AND DENTIN FOR 15 AND 60 SECONDS: AN IN‐VITRO STUDY

Narendra Parihar1, Manish Pilania 2

1BDS, Jodhpur Dental College General Hospital, Jodhpur 2Intern, Department of Conservative Dentistry and Endodontics, Jodhpur Dental College General Hospital, Jodhpur, Rajasthan, India

Corresponding Authors

1. Narendra Parehar Khanda Falsa, Jalori Gate, Jodhpur. Contact no. (+91)9799108142 Email: [email protected]

2. Manish Pilania G-43, Shastri Nagar, Jodhpur Contact no. (+91)9461055068 Email: [email protected] Access this Article Online

AbstractBackground: The purpose of this study was to investigate the relationship between etching of enamel and dentin with different acids: 37% phosphoric acid, 10% maleic acid and 24% EDTA, at different etch durations of 15 and 60 seconds; so as to analyze the surface characteristics of etched enamel, diameter of the dentinal tubules and the depth of demineralization in the tubules under scanning electron microscope (SEM). Method: Thirty freshly extracted maxillary and mandibular premolars, indicated for orthodontic extraction were selected from patients in the age group of 14 to 21 years. The teeth were randomly divided into 3 groups of 10 teeth each and were etched with 37% phosphoric acid, 24% EDTA gel and 10% maleic acid gel respectively for 15 and 60 seconds. The samples were then split along their long axes, dehydrated and sputtered with palladium gold. The sputtered specimens were examined under scanning electron microscope. Results: Acid etching causes various types of etching patterns in enamel indicating preferential dissolution of the enamel prisms. With 37% phosphoric acid, type 2 etching pattern is seen; with 10% maleic acid, type 1 etching pattern is predominant and etching with 24% EDTA leads to type 4 etching pattern. In dentin, etchants widen the dentinal tubule orifices due to demineralization of peritubular dentin and this demineralization extends deep into the dentinal tubules for varying depths depending upon the type of acid used and time of its application. No statistically significant difference exists in the widening of the dentinal tubule orifices between the group I (37% phosphoric acid) and group III (10% maleic acid) specimens. Conclusion: It can be suggested that 15 seconds etching with a milder acid like 10% maleic acid instead of 37% phosphoric acid is sufficient to obtain adequate bond strength because there is no significant difference within the observational parameters, except for the depth of demineralization in tubules. Additional depth is unnecessary because the adhesive systems cannot penetrate completely into the dentinal tubules, leading to nanoleakage.

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Introduction An ideal restorative material would be the one which chemically bonds to the tooth and has strength comparable to that of the tooth structure. In 1955, Buonocore instituted the use of 85% phosphoric acid solution to cause selective decalcification of tooth structure. This produced microporosities in the enamel, increased the surface area, as well as enhanced wettability of the surface providing an intimate contact between tooth and restoration thereby changing the retention form from mechanical to micromechanical. In 1979, Fusayama introduced and popularized the concept of etching of dentin.3 Stronger acids like phosphoric acid not only decalcify the enamel and dentin

surfaces but also demineralize in depth to a greater extent. The increased depth of demineralization is not essentially required because the adhesive systems are not able to penetrate till the complete depth, leading to nanoleakage and incessant degradation.6 Dentin being a vital tissue and containing more organic content than enamel, requires use of milder acids (10% maleic acid and 24% EDTA) so as to prevent damage to micromorphological structure and preserve the integrity of the collagenous mass as they do not denature it.2

Materials and Methods Thirty intact, caries free maxillary and mandibular premolars extracted due to orthodontic reasons were used. These were extracted in the Department of Orthodontics, Jodhpur Dental College General Hospital, Jodhpur, from patients in the age group of 14 to 21 years. After extraction, the teeth were thoroughly cleaned using pumice slurry, and the roots were sheared off from the crown at the labial cementoenamel junction with a diamond abrasives coated safe sided disc. The teeth were then stored in normal saline at 4ºC. At the start of the study, the buccal surfaces of the premolars were ground wet on 320 grit and 600 grit silicon carbide paper till the dentin was exposed to create a flat surface on the enamel and dentin. The samples were then sectioned in the middle along their long axis through the lingual surface with a diamond abrasive coated safe sided disc 1 – 1.5 mm short of the buccal flat surface. The samples were rinsed thoroughly in normal saline to clear off any debris.

The following materials were used for etching of the specimens:-

* 37% phosphoric acid gel.

* 24% Ethylenediamine tetracetic acid (EDTA) gel.

* 10% maleic acid gel.

The prepared samples were randomly divided into three groups:-

Group I, Group II and Group III.

The group I, II and III were further divided into two subgroups of 5 teeth each.

Ia - 5 specimens to be etched with 37% phosphoric acid gel for 15 seconds.

Ib - 5 specimen to be etched with 37% phosphoric acid gel for 60 seconds.

IIa - 5 specimens to be etched with 24% EDTA gel for 15 seconds.

IIb - 5 specimens to be etched with 24% EDTA gel for 60 seconds.

IIIa - 5 specimens to be etched with 10% maleic acid gel for 15 seconds.

IIIb - 5 specimens to be etched with 10% maleic acid gel for 60 seconds.

The etchant gels were applied on the flat buccal surfaces with the help of a brush, the gel was rinsed off from the teeth with 10ml distilled water for 20 seconds. The specimens were then sectioned into two halves with a sharp chisel and mallet by placing the chisel into the groove which had been prepared along the long axis of the samples. The sectioned specimens were utilized for analyzing the depth of demineralization in dentin by the etchants.

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Photograph 1: Armamentarium used for the study

Photograph 2: Materials used in the study

The samples were transferred to small bottles containing graded concentrations of ethyl alcohol (60% to100%). The samples remained in each alcohol concentration for two hours. The dehydrated samples were removed from 100% ethyl alcohol and were mounted on aluminium stubs and placed in vacuum chamber to desiccate them completely.

Samples were viewed under scanning electron microscope (LEO) at various magnifications.

The magnifications selected were:-

- For enamel - x 3,000

- For dentin - x 2,000

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Photograph 3: Scanning Electron Microscope

The parameters to be observed were:- * The etching patterns on enamel surface. The etching patterns were categorized

according to Galil & Wright’s classification4: Type 1 Preferential dissolution of the

prism cores resulting in a ‘honeycomb’ appearance

Type 2 Etch pattern showing preferential

dissolution of the prism peripheries giving a ‘cobblestone’ appearance leaving prism cores relatively unaffected.

Type 3 A more random etch pattern, areas

of which corresponded to type 1 and

2 pattern together with regions in which the pattern of etching could not be related to prism morphology.

Type 4 Pitted enamel surface. Type 5 Flat smooth surface of enamel

without microirregularities. * Diameter of the widened dentinal tubules * Depth of demineralization in the dentinal

tubules. The diameter of the tubules and the depth of demineralization were measured in µm (µ)

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Statistical Analysis

Using the standard deviation, the student t-test was applied

The probability value (p value) was kept constant at 5% significance

STATISTICAL ANALYSIS FOR THE WIDTH OF LOSS OF

ENAMEL PRISM CORE / PERIPHERY

Intragroup comparison

Mean StandardDeviation

Standard Error

T-value Status p<0.05 significant p> 0.05 insignificant

I Ia 0.39µ 0.0554 0.0248

1.65 Insignificant Ib 0.54µ 0.2040 0.0912

II IIa 0.00 0.00 -- IIb 0.00 0.00

III IIIa 3.30µ 0.9758 0.4364

1.195 Insignificant IIIb 4.29µ 1.5614 0.6983

STATISTICAL ANALYSIS FOR DIAMETER OF THE OPENED DENTINAL TUBULES

Intragroup comparison

Mean StandardDeviation

Standard Error


I Ia 2.99 0.8450 0.3779

1.208 Insignificant Ib 3.87 1.3881 0.6208

II IIa 1.94 0.3837 0.1716

3.543 Significant IIb 2.68 0.2663 0.1191

III IIIa 2.93 0.4168 0.1864 1.626 Insignificant IIIb 3.33 0.3671 0.1642

STATISTICAL ANALYSIS FOR DEPTH OF DEMINERALIZATION IN THE TUBULES

Intragroup comparison Mean

StandardDeviation

Standard Error


I Ia 8.87 1.0405 0.4653 2.532 Significant Ib 11.48 2.0587 0.9207

II IIa 0.00 0.00

10.894 Significant IIb 3.92 0.8050 0.3600

III IIIa 7.09 0.5575 0.2493 2.375 Significant IIIb 8.83 1.5382 0.6879

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Results

All etchants changed the micromorphological appearance of enamel and dentin surfaces. Etching of enamel with 37% phosphoric acid gel resulted in a 'cobblestone' appearance of the etched surfaces due to preferential dissolution of the peripheries of enamel prisms (photographs no. 4 and 5). This type of etching is classified as type 2 etching pattern.4 On dentinal surface, phosphoric acid gel removed the smear layer and caused widening of the dentinal

tubules (photograph no. 6). The widening of the tubule orifices occurs due to demineralization of peritubular dentin. The demineralization extends into the depth of tubules giving a 'funneled' effect (photographs no. 8 and 9). The dentinal surfaces treated for 15 and 60 seconds showed particulate residue. This surface residue is possibly silica which is used to thicken the etching gel (photographs no. 6 and 7).

Effect of 37% Phosphoric Acid Gel on Prepared Specimens for 15 Seconds.

Sample

No. Width of loss of enamel prisms periphery

Diameter of opened dentinal tubules

Depth of demineralization in tubules

1. 0.33µ 2.56µ 7.67µ2. 0.34µ 2.9 µ 8.58µ3. 0.44µ 4.3µ 10.43µ4. 0.38µ 2.03µ 9.26µ 5. 0.45µ 3.15µ 8.4µ

Mean 0.39µ 2.99µ 8.86µ Table: 1

Effect of 37% Phosphoric Acid Gel on Prepared Specimens for 60 Seconds.

Sample

No. Width of

loss of enamel prisms

periphery


Depth of demineralization

in tubules

1. 0.44µ 2.67µ 9.64µ 2. 0.36µ 3.90µ 9.31µ 3. 0.50µ 6.22µ 11.96µ4. 0.89µ 3.16µ 14.34µ5. 0.53µ 3.38µ 12.15µ

Mean 0.54µ 3.87µ 11.48µTable: 2

ETCHING OF ENAMEL WITH 37% PHOSPHORIC ACID

(Type 2 Etching Pattern)

Photograph 4: Etching Time-15 Seconds

Photograph 5: Etching Time- 60 Seconds

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ETCHING OF DENTIN WITH 37% PHOSPHORIC ACID

(Widened Dentinal Tubule Orifices)

(Particulate Residue on Surface)

Photograph 6: Etching Time- 15 Seconds

Photograph 7: Etching Time- 60Seconds

ETCHING OF DENTIN WITH 37% PHOSPHORIC ACID GEL

(Depth of Demineralization in Tubules)

Photograph 8: Etching time - 15 seconds


Effect of 24% EDTA gel on enamel after 15 and 60 seconds etching was comparatively insufficient. The surface micromorphology depicted pitted enamel surface which falls into the category of type 4 etching pattern (photographs no. 10 and 11). The dentinal surfaces after 15 seconds and 60 seconds etching were smooth and debris free. The dentinal micrmorphology showed presence of peritubular dentin around the tubule openings in both the subgroups etched with EDTA, indicating its partial

or selective demineralization (photographs no. 12 and 13).

However no discernible depth of demineralization was observed in the specimens.

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Effect Of 24% EDTA Gel for 15 Seconds Application on Prepared Specimens.

Sample

No. Width of

loss of enamel prisms core /

periphery



in tubules

1. 0.00 1.73µ 0.00 2. 0.00 2.54µ 0.003. 0.00 1.52µ 0.004. 0.00 2.01µ 0.005. 0.00 1.88µ 0.00

Mean 0.00 1.94µ 0.00Table : 3

Effect Of 24% EDTA Gel for 60 Seconds Application

on Prepared Specimens

Sample No.

Width of loss of enamel prisms core /

periphery



in tubules

1. 0.00 2.76µ 3.7µ2. 0.00 2.59µ 5.14µ3. 0.00 2.29µ 3.85µ 4. 0.00 3.02µ 2.9µ 5. 0.00 2.72µ 4.02µ

Mean 0.00 2.67µ 3.92µTable : 4

ETCHING OF ENAMEL WITH 24% EDTA GEL (Type 4 Etching Pattern)



ETCHING OF DENTIN WITH 24% EDTA GEL

(Open Dentinal Tubules)



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ETCHING OF DENTIN WITH 24% EDTA GEL (Depth of Demineralization in Tubules)



With 10% maleic acid gel, preferential loss of the prism cores was evident. The surface topography of the etched enamel resembled that of a 'honeycomb' (photographs no. 16 and 17) and is categorized as type 1 etching pattern (according to Galil and Wright's Classification). This study also showed that acid etching with 10% maleic acid for 15 and 60 seconds removed the smear layer and widened the

dentinal tubule orifices. The surface morphology was that of a smooth, debris free surface (photographs no. 18 and 19)

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EFFECT OF 10% MALEIC ACID GEL APPLIED FOR

15 SECONDS ON PREPARED SPECIMENS.

Sample No.

Width of loss of enamel prisms core.



in tubules

1. 3.56µ 2.62µ 7.32µ2. 3.67µ 3.41µ 6.78µ3. 2.19µ 2.57µ 7.04µ 4. 4.61µ 3.35µ 7.90µ 5. 2.49µ 2.68µ 6.43µ

Mean 3.30µ 2.93µ 7.09µTable: 5

EFFECT OF 10% MALEIC ACID GEL APPLIED FOR

60 SECONDS ON PREPARED SPECIMENS

Sample No.

Width of loss of enamel

rods periphery



in tubules

1. 2.45µ 3.52µ 6.91µ2. 5.2µ 2.99µ 11.02µ3. 4.76µ 3.8µ 8.02µ4. 6.13µ 2.93µ 9.4µ5. 2.9µ 3.41µ 8.81µ

Mean 4.29µ 3.33µ 8.83µ Table: 6

ETCHING OF ENAMEL WITH 10 % MALEIC

ACID GEL (Type 1 Etching Pattern)



ETCHING OF DENTIN WITH 10 % MALEIC ACID GEL

(Widened Dentinal Tubule Orifices)


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ETCHING OF DENTIN WITH 10% MALEIC ACID GEL

(Depth of Demineralization in Tubules)



Discussion

The objective of etching enamel is to create microporosities for resin penetration. Dentinal etching removes the smear layer and smear plugs along with demineralization of the surface. It also exposes both intertubular and peritubular collagen.5 Etching of dentin is comparatively a complex phenomenon. This can be attributed to the heterogenous structure of dentin and the dentinal fluid flow in an outward direction that make reliable

bonding to dentin remarkably problematic. The higher organic content of dentin predisposes it to denaturation and weakening of the collagenous mass. Phosphoric acid gel is a strong acid with pH = 1 and dissociation constant, pKa = 2.1. Maleic acid gel is an organic acid with higher molecular weight, pH = 2.9 and dissociation constant pKa = 1.8. EDTA is a powerful organic chelating agent. It is odorless, crystalline white powder with pKa = 6.1 and neutral pH. The results of the study indicate that in group I, in which the prepared surfaces of the specimens were etched with 37% phosphoric acid for 15 and 60 seconds had no significant difference on the etching

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effect and subsequent bond strength values on enamel. On etching of enamel with 10% maleic acid for 15 seconds etching time, the dissolution of the prism cores was lesser, amounting to 3.3µ and with 60 seconds etching time, there was greater dissolution of the prism cores (4.29µ). However, there was no significant difference (p>0.05) in the width of loss of prism core when the teeth were etched for 15 and 60 seconds.24% EDTA etchant gave relatively smooth appearance on enamel and did not alter the intact enamel surface significantly with non-uniform shallow pittings being visible. Photomicrographs of the dentinal surfaces treated with 37% phosphoric acid gel showed that the tubule orifices were open. With 24% EDTA gel, the mean diameter of the tubule orifices was 1.94µ and 2.67µ with 15 seconds and 60 seconds etching respectively. There was no significant difference (p>0.05) in widening of the tubuli when the etching time was extended from 15 seconds to 60 seconds.

Etching with 10% maleic acid gel showed enhanced widening of the dentinal tubules. The mean diameter of opened tubules was 2.93µ when the specimens were etched for 15 seconds and 3.33µ with 60 seconds etching time. No statistically significant difference (p>0.05) was observed. The dentinal surface appeared smooth and free of debris in contrast to the samples etched with 37% phosphoric acid which showed evidence of particulate residue (Silica dioxide) on surface. Intergroup comparison of different acids showed that there exists no significant difference in tubule diameter whether the teeth were etched with 37% phosphoric acid gel or 10% maleic acid gel (p>0.05). However, significant variation exists when the tabulated results were compared between phosphoric acid and EDTA, and maleic acid & EDTA (p<0.05) with EDTA showing less widening of the tubule orifices as compared to the effect of the other two acids. Maximum penetration of the acid into the tubules was seen with 37% phosphoric acid. Phosphoric acid not only dissolves the mineral phase of dentin but also results in denaturation of the collagenous matrix1, 2 which may interfere with hybrid layer. For better retention of the restorative materials, the number of resin tags into dentin is more important than the depth of the tags.

The depth of demineralized dentin to the extent of 11.96µ, 12.15µ and 14.34µ in group etched with 37% phosphoric acid for 60 seconds is not desirable.

Moreover, the adhesive resins may not be able to infiltrate demineralized dentin completely till the depth of demineralization. The incomplete penetration of the demineralized collagen network could result in a delicate zone inside the hybrid layer and, between the hybrid layer and the unaltered dentin that could be susceptible to continuous degradation and a pathway for nanoleakage causing failure of the restoration.6 Further, as the resin penetrates deeper into dentin, the more difficult it will be for them to polymerize if they rely on light activation. In addition, dentinal fluid may interfere with polymerization of the resin tags.7Intergroup comparison between 37% phosphoric acid, 24% EDTA and 10% maleic acid shows comparable results of phosphoric acid and maleic acid apart from the depth of demineralized dentin which is significantly higher (p<0.05) for the 37% phosphoric acid gel group. Considering that 10% maleic acid gel provides clinically acceptable depth of demineralized dentin, 7.09µ (15 seconds) and 8.83µ (60 seconds etch time) it can be inferred that milder acid like 10% maleic acid can replace stronger, more destructive, 37% phosphoric acid for etching of teeth so as to enhance retention of the adhesive restorations.Although acid etching is more than 57 years old, answers to some of the basic questions concerning it are still being sought.

Conclusion With this study we conclude that:-

1. Acid etching causes various types of etching patterns in enamel indicating preferential dissolution of the enamel prisms. With 37% phosphoric acid, type 2 etching pattern is seen; with 10% maleic acid, type 1 etching pattern is predominant and etching with 24% EDTA leads to type 4 etching pattern.

2. In dentin, etchants widen the dentinal tubule orifices due to demineralization of peritubular dentin and this demineralization extends deep into the dentinal tubules for varying depths depending upon the type of acid used and time of its application. No statistically significant difference exists in the widening of the dentinal tubule orifices between the group I (37% phosphoric acid) and group III (10% maleic acid) specimens.

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37% phosphoric acid causes maximum demineralization in the tubules with mean depth of demineralization being more pronounced in 60 seconds subgroup (11.48µ). 10% maleic acid also causes demineralization in tubules but the mean depth is significantly less (8.83µ) than that seen with 37% phosphoric acid. With 24% EDTA etching for 15 seconds, no discernible demineralization is seen in tubules and for 60 seconds.

3. Milder acids like 10% maleic acid can be effectively used for etching of teeth instead of 37% phosphoric acid.

However, a larger sample size is required before deriving any definite conclusions.

Acknowledgement We place on records our deep gratitude towards our mentor, an eminent academician, Dr. (Mrs.) Sumita Kaswan, MDS, Professor, Department of Conservative Dentistry & Endodontics, Jodhpur Dental College General Hospital, Jodhpur, who prudently provided us the impetus for the present study. Photomicrograph Courtesy- Scanning Electron Microscopy Department, AIIMS.

References 1. Blomlof JPS et al. Acid conditioning

combined with single component and two component dentin bonding agents. Q.I. 2001; 32: 711 – 715.

2. Blomlof JPS, Cederlund AL, Blomlof LB et al. A new concept for etching in restorative dentistry. Int J Perio Rest Dent 1999; 19: 31 – 35.

3. Fusayama T, Nakamura M et al. Non pressure adhesion of a new adhesive restorative resin. J Dent Res 1979; 58 (4) : 1364 – 1370.

4. Galil KA and Wright GZ. Acid etching patterns on buccal surfaces of permanent teeth. Paediatric Dent 1979; 1: 230-234.

5. Matos AB, Palma RG et al. Effects of acid etching on dentin surface : SEM morphological study. Braz Dent J 1997; 8(1) : 35 – 41.

6. Perdigao J, Lopes M. The effect of etching time on dentin demineralization. Q.I. 2001; 32 : 19 –

7. Sidhu GK. The effect of acid etched dentin on marginal seal. Q.I. 1994; 25 : 797 – 800.

______________End of artic le___________

SEM EVALUATION OF EFFECT OF 37% PHOSPHORIC ACID GEL, … · EDTA gel for 60 seconds. IIIa - 5 specimens to be etched with 10% maleic acid gel for 15 seconds. IIIb - 5 specimens to

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