Evaluation of biological, physical and chemical

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Original Research Evaluation of biological, physical and chemical properties of mineral trioxide aggregate mixed with 4-META/MMA-TBB Rudra Kaul, Riyaz Farooq, Vibhuti Kaul1, Altaf H Malik2, Aamir Rashid Purra, Lateef Ahmad3 Departments of Conservative Dentistry and Endodontics, 1 Oral Medicine and Radiology, 2 Oral and Maxillofacial Surgery, 3 General Pathology, Government Dental College, Srinagar, Jammu and Kashmir, India

Received : 11-05-12 Review completed : 06-09-12 Accepted : 31-01-13

ABSTRACT Aim: To evaluate the change in physical, chemical and biological properties when mineral trioxide aggregate (MTA) is mixed with a resin 4-methacryloxyethyl trimellitate anhydride (4-META)/methyl methacrylate-tri-n-butyl-borane (MMA)-TBB. Materials and Methods: For biological evaluation MTA was inoculated in Wistar rat’s subcutaneous tissue and peripheral tissue response was checked after 72 h, 7 days, 15 days and 30 days. Setting time was evaluated using Gillmore needle. The Ca++ release at the end of 24 h was checked using ethylenediaminetetraacetic acid titration method. For all the trials MTA mixed with water was kept as a control and the ratio of MTA with resin was 1:1 by weight. Results: The biological reaction was verified by two observers and their readings were matched using kappa test and there was an excellent relevance. There was no significant difference in the tissue reaction at the end of 30 days where both the groups seemed to show healing. Setting time of MTA with 4-META/MMA-TBB was coming to a mean of 26 min (approx.), which is almost 6 times lesser than that of MTA with water. After applying t test, the difference in Ca++ release was found significant (P = 0.00), with mean of 0.044 and 0.031 mol/L of MTA with water and MTA with 4-META/MMA-TBB respectively. Conclusion: Under the parameters of this study, this new experimental cement has better handling, physical and chemical properties. Even its subcutaneous tissue reaction is comparable to MTA mixed with water. Key words: 4-methacryloxyethyl trimellitate anhydride, advancements in mineral trioxide aggregate, mineral trioxide aggregate

Mineral trioxide aggregate (MTA) was studied in a series of investigations, in vivo and in vitro. They reported good sealing ability[1,2] and tissue healing.[3-5] Formation of new cementum over the material was reported in repair of experimentally perforated furcations, [6] after root end filling[1,7,8] and root canal filling of dogs’ teeth.[9] Bridge-like dentin was observed in cases of pulp capping[5,10,11] and pulpotomy[3,12] in monkey and dog teeth. On the other hand, 4-methacryloxyethyl trimellitate anhydride (4-META)/methyl methacrylate-tri-n-butylborane (MMA)-TBB, a light-cured opaque resin, was Address for correspondence: Dr. Rudra Kaul E-mail: [email protected] Access this article online Quick Response Code:

Website: www.ijdr.in PMID: *** DOI: 10.4103/0970-9290.118381

Indian Journal of Dental Research, 24(4), 2013

prepared with 4-methacryloxyethyl trimellitate anhydride 4-META, bifunctional methacrylates and titanium dioxide with MMA added to serve as a solvent of 4-META[13] introduced by Nakabayashi et al. in 1988. This resin has been used as a pulp capping agent and no cytotoxic response has been noticed.[14] Water when mixed with MTA causes the hydration of anhydrous mineral oxide compounds via the dissolution of the compounds, followed by the crystallization of hydrates. The reason for the various biological activities of MTA is Ca++ ions which are continuously released from calcium hydroxide, maintaining high pH. This dissolution of calcium hydroxide may negatively influence the physical properties of MTA in a longer run. Scanning Electron Microscope analysis of water-immersed MTA revealed an increased porosity, which may have been caused by the dissolution of calcium hydroxide and other hydration products. It has been confirmed that high amounts of Ca++ in a cell culture environment might down-regulate cell proliferation.[15] Another problem is the poor handling properties, very short working time and prolonged setting time.[16] MTA is prepared by mixing its powder with sterile water in a 3:1 powder-to-liquid ratio.[8] The mean setting time of MTA is 418


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Properties of MTA mixed with 4-META/MMA-TBB

165 min, which is longer than amalgam, Super epoxy benzoic acid (EBA) and intermediate restorative material (IRM).[17] The idea behind mixing MTA with a resin, i.e., 4-META/ MMA-TBB was to obtain a cohesive mass, which had significantly lesser setting time but at the same time should be tolerated by the tissues and not totally bind the Ca++ in the mix, as Ca++ forms the basis of biological influence of MTA. On this basis, it was decided to evaluate the change in physical, chemical and biological properties of MTA when mixed with 4-META/MMA-TBB.

MATERIALS AND METHODS For all the experiments MTA Angelus (Angelus, Londrina, PR, Brazil) was used. MTA: Water = 3:1 was kept as control and MTA: 4-META/MMA-TBB in a ratio of 1:1 by weight was the experimental cement.

Biological evaluation

This study was carried out following the guidelines of the Ethics Committee for Teaching and Research in Animals, Government Medical College, Srinagar. Twenty four male Wistar rats, weighing about 250 g each, from the Central Animal Laboratory of RRL, Jammu, were randomly divided into 2 groups. Each group was subdivided into 4 subgroups; every subgroup was allotted 3 rats used for four different experimental periods of 72 h, 7 days, 15 days and 30 days. Group 1, in all its subgroups, received implants containing MTA: Water = 3:1. Similarly, Group 2 used mixture of MTA with 4-META/ MMA-TBB in a ratio of 1:1 by weight. The materials were mixed at the moment of implantation.

Preparation of animals and procedures for implantation

The same surgical sequence was used for all animals. The animals were anesthetized with an intramuscular injection of Xylocaine 0.5 mL/kg body weight. Under aseptic precautions, two straight incisions of approximately 1.5 cm were made with a No. 10 Bard-Parker blade exposing the subcutaneous connective tissue. Twenty four polyethylene tubes (5 mm in diameter and 1 cm in length) were disinfected in 5% sodium hypochlorite for 5 min and then washed in deionized water for 15 min under sonification. Half of the tubes were filled with one of the cements to be tested and the other half was filled with MTA mixed with water as control and inoculated into the animals’ back. The wounds were then sutured. All animals received a normal diet and water ad libitum throughout the entire study. The animals were anesthetized and a sample of the tissue containing the implants was removed at 72 h, 7 days, 15 days and 30 days after implantation. The specimens were fixed 419

in buffered 10% formalin for 24 h. After histotechnical processing, 5 µm thick sections were taken and stained with H and E.

Microscopic analysis

For the evaluation of the chronic inflammatory reaction of subcutaneous connective tissue, necrotic, granulomatous tissue, presence of macrophages, multinucleated giant cells, lymphocytes, granulation or fibrous tissue were observed and classified as absent, discrete, moderate and intense by two observers previously calibrated, in relation to numerical scores of 0, 1, 2 and 3, respectively. One amongst these reviewers, one was made the prime observer and the readings were matched with that of the other for its relevance using kappa test.

Chemical evaluation

T o c a l c u l a t e t h e m o l a r c o n c e n t r a t i o n o f C a ++, ethylenediaminetetraacetic acid (EDTA) titration or complexometric titration method was used. For chemical analysis, 10 polyethylene tubes (5 mm in diameter and 1 cm in length) were filled with MTA (5 experimental and 5 control) and immersed in distilled deionized water (5 ml) for 24 h in separate test tubes. After 24 h, the polyethylene tubes were removed. A blue dye called Patton and Reeder’s indicator (PR) was used as an indicator. This blue dye also forms a complex with the Ca++, changing colour from blue to pink/red in the process, but the dye–metal ion complex is less stable than the EDTA–metal ion complex. As a result, when the Ca++-PR complex is titrated with EDTA, the Ca++ reacts to form a stronger complex with the EDTA. A preset EDTA 0.025 mol/L solution was made. For the titration, the indicator was added to the sample solution containing the Ca++ forming the pink/red calcium ionindicator complex (Ca++-PR). This solution was then titrated with EDTA. The endpoint was considered to occur when the solution turned blue, indicating the Ca++-PR complex to be completely replaced by the Ca++-EDTA complex with the PR indicator reverting to its former blue color. For results, the average volume of EDTA solution used from concordant titers was noted, which lead to calculation of the moles of EDTA required to complex the Ca++ in the sample. Using the method ratio of Ca++: EDTA = 1:1, total concentration of Ca++ in mol/L was obtained. Later the results were verified using t test.

Physical evaluation

To check for any change in the setting time of experimental cement, the cement was tested using Gillmore needle for its initial setting time. For this, the experimental cement was filled up to 1 cm height in a small ceramic vessel having a diameter of 2 cm. At an interval of every 3 min the setting time was tested till the needle did not penetrate. Three trials were done on both the cements and the mean was calculated. Indian Journal of Dental Research, 24(4), 2013


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Properties of MTA mixed with 4-META/MMA-TBB Table 1: Grading for biological analysis Duration Reaction

a

b

72 h

7 days

15 days

30 days

H20 Resin H20 Resin H20 Resin H20 Resin

Necrosis Granulomatous Macrophages Giant cells Lymphocytes Repair Fibrosis

0 0 0 0 1 0 0

0 0 1 0 1 0 0

0 0 1 0 2 0 0

1 0 1 0 1 0 0

0 1 2 0 1 1 1

0 1 1 0 2 1 2

0 0 0 0 1 3 1

0 0 0 0 1 2 2

Table 2: mol/L Ca+ + release after 24 h

c

d

H 2O

Resin

0.045 0.045 0.047 0.042 0.042

0.033 0.037 0.03 0.027 0.03

After applying t test, the difference in Ca++ release was found to be significant (P = 0.00) with a mean value of 0.044 mol/L and 0.031 mol/L for control and experimental cement respectively. Thus, mixture of MTA with resin released significantly less Ca++ than MTA with water over a period of 24 h. e

f

Physical analysis

The different time periods recorded in 3 different trials for initial setting time for experimental cement were recorded [Figure 2 and Table 3].

g

h

Figure 1: (a) (Mineral trioxide aggregate [MTA] with water after 72 h) Dense neutrophilic infiltration; (b) (MTA with resin after 72 h) More intense acute inflammation with deeper involvement of tissues; (c) (MTA with water after 7 days) A mixed chronic and acute inflammation seen in deeper tissues; (d) (MTA with resin after 7 days) intense mixed inflammation, with hints of necrosis; (e) (MTA with water after 15 days) Granulomatous change with persisting inflammation and evident fibroblast; (f) (MTA with resin after 15 days) Lesser granulomatous change, fewer fibroblasts and intense inflammation; (g) (MTA with water after 30 days) Almost resolved inflammation showing healing and hints of perivascular inflammatory cells; (h) (MTA with resin after 30 days) Hints of inflammation seen in deeper tissues

RESULTS AND DATA ANALYSIS Biological analysis

The relevance of the readings given by the prime observer when compared with the readings of the second one showed 96% reliability when subjected to kappa test. So the readings of primary observer were taken as valid [Figure 1 and Table 1].

Chemical analysis

The mol/L Ca++ release by both MTA with resin and MTA with water after being immersed in distilled water for 24 h was measured [Table 2]. Indian Journal of Dental Research, 24(4), 2013

Obtaining a mean of 26 min for the experimental cement and 161 min (approx.) for control showed the MTA with resin cement to set 6 times faster than that of MTA with water.

DISCUSSION MTA has been shown to be a very biocompatible material, in fact more biocompatible than Super EBA and IRM.[18] There are certain short comings regarding high setting time and cumbersome handling.[16] The idea of the study was to mix MTA with 4-META/MMA-TBB to form a cement which had better handling properties with lesser setting time. A similar experiment was performed using epoxy resin, but the cement was just evaluated for its biological properties only and the material was found to instigate similar response as MTA with water.[19] In another study conducted on 4-META assessing its effect on pulp, it was concluded that the resin is slightly cytotoxic but does not hamper cell proliferation or differentiation, thus, promoting its use as a pulp capping agent.[14] In this experiment, after MTA was mixed with 4-META/ MMA-TBB, an excellent cohesive mass which had far better handling properties and 6 times lesser setting time than MTA with water was achieved. Both these findings were a boon as far as the clinical performance of MTA is concerned. 420


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Figure 2: Setting time under Gillmore needle for mineral trioxide aggregate mixed with 4-methacryloxyethyl trimellitate anhydride

Table 3: Initial setting time in 3 different trials MTA + Resin 27 min and 21 s 25 min and 16 s 25 min and 31 s

MTA + Water 157 min and 30 s 164 min and 7 s 161 min and 12 s

MTA = Mineral trioxide aggregate

This venture could have contributed to lesser biocompatibility, which was not seen in the animal subcutaneous inoculation. Moreover, the inflammatory changes had almost resolved in both the groups after 30 days. There were certain areas of perivascular inflammatory cells seen in the resin group. Water group showed very few inflammatory cells in deep tissues and higher repair rate. As no giant cells were seen in both the groups, so no evidence of any profound chronic inflammation was found. Ca++ release forms the basis of biological properties of MTA[20] and resin, being insoluble in aqueous fluids could have trapped all the calcium, leaving the cement biologically inactive. Furthermore, the loss of excessive Ca++ which renders the cement weak was also checked by the resin. Maintaining a check on Ca++ release can help strike a balance between the healing properties of MTA and the cement strength. Further research on this material evaluating its compressive strength, analyzing the structure of the set cement, bond strength with dentin, optimum Ca++ release levels for MTA to display biological properties and compatibility using immunological assays before it can be used in humans is needed.

CONCLUSION As per the parameters of this study, MTA was mixed with a resin 4-META/MMA-TBB and evaluated for biological compatibility, physical and chemical properties. The 421

experimental cement showed almost equal subcutaneous tissue response, almost 6 times lesser setting time and only 75% of calcium release after 24 h as compared to MTA mixed with water.

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18. Fernández-Yáñez Sánchez A, Leco-Berrocal MI, Martínez-González JM. Metaanalysis of filler materials in periapical surgery. Med Oral Patol Oral Cir Bucal 2008;13:E180-5. 19. Menezes R, de Moraes FG, Oliveira RC. Subcutaneous tissue reactions to MTA, portland cement and experimental cement. Rev de Clín Pesq Odontol 2005;1:11-3. 20. Bakland LK, Andreasen JO. Will mineral trioxide aggregate replace calcium hydroxide in treating pulpal and periodontal healing complications subsequent to dental trauma? A review. Dent Traumatol 2012;28:25-32. How to cite this article: Kaul R, Farooq R, Kaul V, Malik AH, Purra AR, Ahmad L. Evaluation of biological, physical and chemical properties of mineral trioxide aggregate mixed with 4-META/MMA-TBB. Indian J Dent Res 2013;24:418-22. Source of Support: Nil, Conflict of Interest: None declared.

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