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Year : 2017  |  Volume : 7  |  Issue : 1  |  Page : 23-28

An in vitro study to evaluate the demineralizing effect of maleic acid and cell attachment on cemental surface

1 Department of Periodontology, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka, India
2 Department of Conservative Dentistry and Endodontics, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka, India

Date of Web Publication10-Jan-2017

Correspondence Address:
Jothi Varghese
Department of Periodontology, Manipal College of Dental Sciences, Manipal University, Manipal, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1658-5984.197988

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Introduction: Root biomodifying agents are adjuncts to periodontal regeneration.
Aim: This study evaluates the demineralization effect of a chelating agent, maleic acid (MA) when used as a root biomodifier on the cemental surface.
Materials and Methods: Thirty single-rooted anterior teeth were selected for this study. The teeth were decoronated and split longitudinally to form sixty radicular halves. They were randomly assigned to four groups of 15 fragments each according to the demineralizing agents used and treatment time of 1 and 3 min. Group 1 treated with 50% citric acid (CA) solution at pH 1, Group 2 treated with 17% ethylenediaminetetraacetic acid (EDTA) solution at pH 7. Group 3 treated with 7% MA at pH 1.3 and Group 4 treated with saline. The specimens were then prepared to view under scanning electron microscopy (SEM). To study the adherence of cells on the root surface, human periodontal ligament cells were cultured and were placed in plates containing root halves, and cell adherence was viewed using SEM. The data were statistically analyzed by Pearson's Chi-square test (χ2) to evaluate the effect of the each test agent. A significance level was set at P< 0.05.
Results: All the three test agents were successful in eliminating smear layer at both time intervals. Intergroup comparison at both time intervals demonstrated, MA to have a better smear layer removal ability than CA and EDTA.
Conclusion: This particular study provided preliminary evidence on the demineralizing effect of 7% MA when used as a root conditioning agent.

Keywords: Cell attachment, citric acid, ethylenediaminetetraacetic acid, maleic acid, root biomodification

How to cite this article:
Varghese J, Ballal NV. An in vitro study to evaluate the demineralizing effect of maleic acid and cell attachment on cemental surface. Saudi Endod J 2017;7:23-8

How to cite this URL:
Varghese J, Ballal NV. An in vitro study to evaluate the demineralizing effect of maleic acid and cell attachment on cemental surface. Saudi Endod J [serial online] 2017 [cited 2020 Feb 27];7:23-8. Available from: http://www.saudiendodj.com/text.asp?2017/7/1/23/197988

  Introduction Top

The revival of the lost periodontium using therapeutic measures has always been the clinician's unceasing objective since decades. Periodontal infection has been regarded as the prime factor for inhibiting a substantial regeneration. Such a condition could contribute to the paucity of a biologically compatible root surface which is inherent for an epithelial and connective tissue cell attachment.[1],[2] To achieve considerable amount of periodontal regeneration, the diseased root surface should be devoid of local and cytotoxic substances.[3],[4] This desirable prerequisite is carried out by mechanical instrumentation (scaling and root planing). Further, this situation could result in possibilities for exposure of dentinal tubules thereby creating avenues for which could further contribute to inflammatory lesions in the pulp [5],[6] and/or create routes for the dissemination of bacterial toxins causing localized inflammatory pulpal response.[7] In case of lesions affecting the pulp or periodontium, the sequence of management should be treating the primary disease. In general, initiation of endodontic treatment warrants the cementum layer to be intact as it resolves infection within the root canal and avoid further exposure of dentin and improves periodontal healing.[5],[8]

Furthermore, mechanical instrumentation results in the formation of a smear layer of organic and mineralized debris on the root surface. This adherent layer creates a physical barrier, inhibiting new attachment, and acting as a substrate for bacterial growth creating various structural and biochemical changes on the periodontitis-affected root surfaces.[9],[10]

Historically, root conditioning using citric acid (CA) was performed during regenerative periodontal therapy that aided to eliminate microbial endotoxins, anaerobic bacteria, and expose collagen bundles which served as matrix for new connective tissue attachment to cementum. This resulted in effectual treatment amenable for favorable healing [11] and elimination of smear layer also ensued pulpal protection.[12]

Ethylenediaminetetraacetic acid (EDTA) owing to the neutral pH and its ability to etch and expose collagen matrix on root surface makes it a favorable substrate for fibroblast attachment.[13],[14] An important parameter that denotes the success in adhesive dentistry is the bonding system which is employed by the use of organic acids. One such, mild acid which has been used to etch the dentinal surface is maleic acid (MA).[15] Further elucidative studies were performed in the field of endodontics which suggested its beneficial effect as a final root canal irrigant.[16],[17],[18]

To the best of our knowledge, there is no study conducted using MA as a root biomodifier. Hence, the aim of this study was to evaluate the demineralizing effect of MA and compare it with two other commonly used root conditioning agents.

  Materials and Methods Top

A total of thirty single rooted human permanent maxillary anterior teeth extracted only due to advanced periodontal disease were selected for this study. Ethical clearance was taken from the Institutional Review Board (IEC 168/2015, Manipal University). Following extraction, the teeth were washed with saline so as to eliminate adherent soft tissues and hand instruments were used so as to remove calculus. The specimens were stored in 10% formalin. The cemental surface was meticulously root planed and irrigated with distilled water so as to obtain a smooth, clean surface. The teeth were decoronated and split longitudinally to form sixty radicular halves. Each half was further cut to remove the coronal and apical third of the root so as to preserve the mid-root region. The fragments were designed into a rectangular shape (approximate dimensions of 5 mm × 3 mm × 1 mm) using low-speed diamond bur (Horico, Germany) under water irrigation.

The root slices were then sterilized to eliminate further contamination.[19] Following which, the root halves were randomly assigned to four groups of 15 fragments each according to the demineralizing agents used and treatment time. Group 1 CA: Treated with 50% CA solution (KMC Pharmacy, Manipal, India) at pH 1, Group 2 EDTA: Treated with 17% EDTA solution (Merck, Darmstadt, Germany) at pH 7. Group 3 (MA): Treated with 7% MA (KMC Pharmacy, Manipal, India) at pH 1.3 and Group 4 (control) treated with saline.

All the specimens in each group were burnished with the respective demineralizing agent using sterile cotton pellet for time intervals of 1 and 3 min. Following which, all the specimens were washed with distilled water.

Scanning electron microscopy preparation of specimens

The specimens were then fixed in 2% glutaraldehyde for 15 min and then washed with phosphate buffer solution (PBS) and further postfixed for 12 h at 4–6°C with 1% (wt/vol) osmium tetroxide in 0.1M PBS buffer for 30 min at 37°C and again 0.1M PBS was used as a final wash for around 10 min. The radicular fragments were then dehydrated in varying concentrations of ethyl alcohol (70%, 80%, 90%, 95%, and 100%) for 10 min. The specimens were dried using a SAMDRI PVT-3 critical point dryer apparatus (Tousimis Research Corp., Rockville, MD) using liquid CO2 replacement. The dried samples were gold sputtered and examined under scanning electron microscopy (SEM) (LEO 440I, Carl Zeiss, Tokyo, Japan) to evaluate for the changes on the demineralized root surfaces. Several photomicrographs were taken to observe the morphological changes on the surface.

The images were scored according to the criteria suggested by Torabinejad et al.,[20] 1 = no smear layer (no smear layer on the surface of root canal; all tubules were clean and open), 2 = moderate smear layer (no smear layer on the surface of root canal, but tubules contained debris), and 3 = heavy smear layer (smear layer covered the root canal surface and the tubules).

Cell culture and growth

To observe the attachment of fibroblast on the root surface conditioned with the demineralizing agents, human periodontal ligament cell line was obtained from teeth that were freshly extracted. The tooth was directly transferred into a conical bottle containing 10% of fresh fetal bovine serum (Gibco, Invitrogen, Grand Island, NY, USA) and immediately carried into laminar flow hood, wherein the crown of tooth was carefully grasped by means of a sterile forceps so as to avoid direct contact with root surface. The tooth was then washed thrice with 1 ml of PBS and 0.5 ml of antibiotic solution (mixture of 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% amphotericin B).[21] A sterile bard parker no. 15 was used to scrape the periodontal tissue from the mid portion of the root surface into a petri dish. Mid root was chosen so as to avoid bacterial contamination from the gingival tissue (coronal third) or pulp tissue (apical third). The tissues pieces were then placed individually into the 6-well plate and after 15 min, 2 ml of Dulbecco's modified Eagle's medium (DMEM) was carefully added into each well plate so that the tissue fragments are immersed completely. The petri dishes were then placed in an incubator at 37°C in an atmosphere of 5% CO2, which continued for 5 days until confluence was noticed. At this stage, the cells were ready to be trypsinized for the establishment of subcultures. The radicular samples were then cut into smaller dimensions so as to fit the well plate containing the culture medium (DMEM). Each specimen burnished with the demineralizing agent on the cemental surface was then placed into the well plate with the treated surface facing up. It was then placed in an incubator at 37°C for 24 h. The specimens then fixed in 2% glutaraldehyde (Sigma, St. Louis MO, USA) in PBS for 15 min, following which the same method was performed as described earlier for the preparation of SEM analysis.

The results of this study were analyzed by Pearson's Chi-square test (χ2) to evaluate the effect of the each test agent at different time intervals. A significance level was set at P < 0.05.

  Results Top

The inter examiner's reliability was verified using the kappa test and was found to be statistically insignificant. The comparative percentage of smear layer removed by the test agents on the root surface is demonstrated in [Figure 1]a and [Figure 1]b. There was no significant difference between CA and EDTA group in the removal of smear layer at 1 and 3 min (P > 0.05). However, MA removed smear layer more effectively than CA and EDTA at both 1 and 3 min time interval. In the control (saline), all specimens showed heavy smear layer on the root surface [Figure 1]a and [Figure 1]b.
Figure 1: (a) Demineralizing effect of three different root conditioning agents and control (saline) on 15 samples at 1 min time interval. (b) Demineralizing effect of three different root conditioning agents and control (saline) on 15 samples at 3 min time interval

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All the specimens were checked at 1 and 3 min time interval. All the three test agents were successful in eliminating smear layer at both time intervals. In this study, a scoring criteria were utilized for the quantification of smear layer removal, which will further define the potential of the etchant or chelating agent used. In 1 min time interval, from total of 15 specimens, 9 specimens of the CA group showed no smear layer whereas the remaining 6 specimens demonstrated moderate smear layer. Similarly, in the EDTA group, 5 specimens demonstrated no traces of smear layer, whereas remaining 10 specimens had moderate smear layer present. However, within the MA group, 4 specimens exhibited no smear layer, whereas the remaining 11 specimens showed moderate amount of smear layer.

In the specimens categorized under the 3 min time interval, a similar pattern of smear layer removal was seen in the CA and EDTA group. But within the MA group, 12 specimens showed no smear layer, whereas remaining 3 had moderate smear layer present. All the 15 samples in the control group at both time intervals (1 min, 3 min) exhibited moderate to heavy smear layer [Figure 1]a and [Figure 1]b. The SEM photomicrographs [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d,[Figure 2]e,[Figure 2]f,[Figure 2]g,[Figure 2]h reveal the smear layer removal ability of the three root conditioning agents through the presence of patent dentinal tubules on the root dentin surface.
Figure 2: Panel of scanning electron microscopy photomicrographs. (a-d) A representative specimen of citric acid (Group 1), ethylenediaminetetraacetic acid (Group 2), maleic acid (Group 3), and control (Group 4) at 1 min time interval. (e-h) Specimens of citric acid (Group 1), ethylenediaminetetraacetic acid (Group 2), maleic acid (Group 3), and control (Group 4) at 3 min interval

Click here to view

Intergroup comparison at both time intervals demonstrated, MA to have a better smear layer removal ability than CA and EDTA [Figure 1]a, [Figure 1]b, and [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d,[Figure 2]e,[Figure 2]f,[Figure 2]g,[Figure 2]h. However, between the groups CA and EDTA, the group CA showed better demineralizing effect than EDTA but was not statistically significant. On the contrary, comparing CA and MA groups, the demineralizing effect of MA was found to have a better than CA but lacking statistical significance [Figure 1]a and [Figure 1]b, and [Figure 2]c and [Figure 2]g.

Cell attachment

[Figure 3]a,[Figure 3]b,[Figure 3]c,[Figure 3]d shows SEM photomicrographs depicting the attached cells on the surface of root fragments belonging to the experimental groups compared to the control group.
Figure 3: Scanning electron microscopy photomicrographs of periodontal fibroblasts attached on root conditioned dentin discs. (a) Citric acid group, (b) ethylenediaminetetraacetic acid group, (c) maleic acid group, and (d) control group

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  Discussion Top

To mimic the clinical management, before the use of demineralizing agents on the root surfaces, mechanical instrumentation was done. This resulted in the formation of smear layer which was confirmed by the control group, in this study [Figure 3]d. Therefore, root conditioning has been used to disinfect and to demineralize the root surface, remove the smear layer, exposing extracellular matrix of mineralized tissues, such as Type I collagen, and facilitating attachment between the root surface and the healing connective tissue.[22]

Hence, the final outcome of the chemically treated process is to create a conducive environment for periodontal regeneration, by exposure of collagen network, and further providing a milieu for fibroblast cell growth and attachment.[23]

The initial studies conducted by Register and Burdick [24] have concluded that CA, (pH 1 for 2–3 min) have resulted in optimum cementogenesis and connective tissue attachment. Bogle et al.[25] proposed that EDTA (12–24% at neutral pH for 3 s to 3 min) aimed at removing smear layer and widening of dentinal tubules without causing damage to biologic structures. Furthermore, the use topical fluoride gel for root conditioning has been proposed in case of replanted avulsed teeth.[26] Hence, the time period chosen for the study was 1 min and 3 min, so as to utilize the beneficial demineralizing effect of the conditioning agents. The method of application plays an important role in exposing collagen fibers to enhance cellular adherence. The technique of application of test agent used was the burnishing technique, which aids in effectual removal of smear layer and exposure of the dentinal tubules as proposed by Sterrett and Murphy.[27]

In this study, the demineralizing effect was noticed by the number of patent dentinal tubules as revealed by the SEM photomicrographs. All the three test agents used in this study removed smear layer and resulted in the exposure of dentinal tubules [Figure 1] and [Figure 2]. This was in accordance with Blomlöf et al.[13] and Ruggeri et al.[14] whose work concluded that the use of CA and EDTA led to the exposure of fibers and proteoglycans without destruction of the dentin matrix. MA was observed to be effective for smear layer removal from an instrumented root canal.[16] Similarly, in this study also it was found to be effective than CA and EDTA, which may be probably due to its high demineralizing efficacy.[28]

Furthermore, there is evidence of chemisorption of MA to enamel and hydroxyapatite. MA can decalcify and chemically adhere to hydroxyapatite. This ionic bonding produces calcium maleate and other products, which could make it a functional component to enhance its effectiveness.[29]

Regardless of the operational limitations, SEM analysis remains as a predictable tool to validate the attachment of the fibroblasts cells to the root surfaces.[22] In this study, SEM demonstrated the presence of cell attached on to the root surface of the experimental groups when compared to control [Figure 3]a,[Figure 3]b,[Figure 3]c. This demonstrates its ability for enhanced periodontal healing process.[30] In an SEM investigation performed by Babay on periodontally involved root surfaces, it was observed that the cultured fibroblasts attached better on rough-surface formed by demineralizing agents compared to only instrumentation.[31] To evaluate the number of attached cells onto the cemental surface, histometric analysis with light microscopy could be used, in which the cell is considered attached if its nucleus is in contact with the surface or lay within the intercellular matrix which was attached to surface.[32] Since this is the first study to use MA as root conditioning agent, the observation of cell attached onto the root surface burnished with 7% MA, supports the concept that it could provide a biocompatible zone for periodontal regeneration [Figure 3]c. Its low-toxicity feature [33] could also warrant its use in vivo.

  Conclusion Top

Within the precincts of this in vitro study, it could be concluded that this particular trial provided preliminary evidence on the demineralizing effect of 7% MA when used as a root conditioning agent. MA removed smear layer effectively compared to EDTA and CA. Considering its beneficial qualities such as low toxicity, chemical adherence to hydroxyapatite, fibroblast attachment, etc., could provide enhanced results in terms of regeneration. However, further studies need to be performed to evaluate the influence of MA on periodontal ligament fibroblasts adherence and viability to validate healing outcome.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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