Journal of Dental Implant Research 2022; 41(2): 15-24
Evaluation of the effect of EDTA on the antifungal properties, flexural strength, and colour stability of heat polymerised PMMA resin for implant overdentures
Yasmin Fathima, Jayakrishnakumar Sampathkumar , Hariharan Ramakrishnan , Nagarasampatti Sivaprakasam Azhagarasan
Ragas Dental College & Hospital, Chennai, India
Correspondence to: Hariharan Ramakrishnan,
Ragas Dental College & Hospital, 2/102, East Coast Road, Uthandi, Chennai 600119, Tamilnadu, India. Tel: +04424530006, Fax: +04452123995, E-mail:
Received: April 11, 2022; Revised: June 7, 2022; Accepted: June 8, 2022; Published online: June 30, 2022.
© The Korean Academy of Implant Dentistry. All rights reserved.

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Purpose: To evaluate the effect of ethylenediaminetetraacetic acid (EDTA) on the antifungal properties, flexural strength, and color stability of heat polymerized polymethyl methacrylate (PMMA) denture base resin.
Materials and Methods: Eighty PMMA heat cure denture base resin test samples of the following sizes were fabricated: 10×1 mm (disc), 64×10×3.3 mm (strip), and 50×0.5±0.05 mm (disc) with or without EDTA. The PMMA heat cure denture base resin samples formed one group (GROUP I, n=40) and PMMA heat cure denture base resin samples incorporated with 8% EDTA were the second group (GROUP II, n=40). These were further subdivided according to the testing parameters as follows: Qualitative analysis of antifungal properties (GROUP I: 10 and GROUP II: 10), quantitative analysis of antifungal properties (GROUP I: 10, GROUP II: 10), flexural strength, and colour stability, 10 samples each for both groups and properties. The antifungal properties of GROUP I and GROUP II were evaluated qualitatively and quantitatively by measuring the zone of inhibition using the disc diffusion method and colony forming units (CFU), respectively. The flexural strength and color stability were evaluated using the three-point bend test and a spectrophotometer. The color difference (ΔE) was calculated.
Results: The mean zone of inhibition for GROUP I and GROUP II samples was 0 mm and 9.2 mm, respectively. The mean CFU of GROUP I and GROUP II was 119.50×106 and 45×, respectively. The mean flexural strength of GROUP I and GROUP II samples was 145.43 MPa and 112.57 MPa. The colour difference between GROUP I and GROUP II samples ΔE was 2.89.
Conclusions: PMMA heat polymerized denture base resin incorporated with EDTA exhibited antifungal properties and a minimum reduction in flexural strength, with a perceptible color difference. This modified PMMA resin is recommended for implant overdentures.
Keywords: Polymethylmethacrylate, Denture base, Acrylic resin, EDTA, Antifungal agent, Colony forming units, Flexural strength

Rehabilitation of partial and completely edentulous arches with removable dental prosthesis is mainly fabricated from Polymethyl methacrylate Heat Cure Denture Base Resin for more than six decades1). Poor denture hygiene is considered a major risk factor in the development of local and systemic infections such as denture stomatitis, cardiovascular complications, and respiratory tract infections. The predominant microorganisms detected on the denture surface include Candida albicans and Candida glabrata2).

Methods available to clean the dentures include mechanical and chemical. However, hypochlorite-based cleansers cause corrosion of metal components in dentures3). Studies have established that ultra-sonic and microwave cleansers predominantly reduced fungal colonization on removable dentures. However, these technologies have limited applicability owing to their excessive cost and ability to damage the denture base material4).

Several studies have shown that denture cleansers exhibit effective Anti-Candida Albicans biofilm activity, both in removal and disinfection, however, residual biofilm retention could lead to regrowth and denture colonization was observed4). Thus, the incidence of relapse of the infection is high. Several investigators attempted to combine antifungals or antiseptics with temporary soft liners or with denture acrylic resin, such as nystatin, miconazole, ketoconazole, fluconazole and itraconazole, chlorhexidine, triclosan, titanium dioxide, and zeolites, however, the efficacy of these combination depends on the release of agents that act on the microbes. One major drawback of this technique is that these agents may exhibit cytotoxicity and can deteriorate the mechanical properties of the resin. Options, such as surface treatment by glow-discharge plasma, glazes, incorporation of polar radicals into the polymer, and the copolymerization of fluoroalkyl methacrylate have also been studied5).

A major limitation of these approaches is that the antifungal drugs incorporated into the PMMA Heat Cure Denture Base Resin may not be able to endure the polymerization process and may negatively affect physical and mechanical properties. Chelating agents such as EDTA has shown to possess antifungal property, as it interrupts microbial biofilm through chelation of iron, calcium, and magnesium from the cell wall of the microorganism and several essential components that are associated with the biofilm matrix. Studies have shown that EDTA possesses increased antifungal activity by limiting growth and nutritional conditions with this mechanism6,7). For quantification of efficacy of surface designs based on the release of antimicrobials, various assays are described which are mainly categorized as “suspension methods”8). Flexural fatigue in PMMA dentures is mainly evidenced by the propagation of midline cracks which is due to the stress concentration around the microcracks formed in the material due to the continuous application of small forces9-11).

The resistance of resins to color changes can be influenced by the structure of the inorganic fillers, the physical properties of the resin, and their chemical characteristics12). According to CIELAB, if the color difference between two objects, is calculated as ΔE, where the clinical acceptability threshold value is ΔE ≤2.25 units. Previous studies1,13-15) have evaluated the antimicrobial property of different synthetic and herbal compounds on both Denture Base Resins and restorative Resins, but studies evaluating the effect of EDTA as an antifungal agent on the mechanical and optical properties of PMMA resin are very sparse.


The study was approved by the Institution review and ethical board. Three custom made metallic molds were fabricated to obtain the disc-shaped wax patterns of size 10×1 mm (Antimicrobial property)strip shaped wax patterns of size 64×10×3.3 mm (Flexural strength) and disc-shaped wax patterns of size 50×0.5 mm (Colour stability) respectively.

Modeling wax was uniformly melted and poured into three custom-made metallic molds and was allowed to solidify. The wax patterns of sizes 10×1 mm, 64×10×3.3 mm, and 50×0.5+0.05 mm were then removed from the custom molds and this process was repeated to prepare eighty wax patterns. The prepared wax patterns were finally measured using a digital caliper to ensure a uniform dimension. Following the manufacturer's instructions, a water/powder ratio of 0.45 was used in preparing the plaster slurry for investing the wax patterns in a denture flask. Upon completion of the final setting of gypsum, the denture flask was immersed in boiling water for 4 minutes for wax elimination. The flask was then removed from the water, and the softened wax was removed. The residual wax sticking to the mold cavity was carefully flushed using boiling water. The mold cavity was then cleaned with mild detergent and rinsed with hotywater. 8% of EDTA powder by weight was measured using a digital balance and was incorporated into the PMMA polymer. Proportioned powders were mixed thoroughly using a vacuum mixer. This mixture was used to prepare PMMA test samples for GROUP II only.

Before the mixing of polymer monomer, the mold cavity was coated with a mold separating medium (cold mold seal) 3:1 by volume of polymer to monomer was used in the preparation of resin dough. Upon reaching the appropriate consistency the dough was packed in the mold cavity and following trial closure the dental flask was kept under pressure to complete bench curing. The flask was then placed in an acrylizer at a constant temperature of 74 degrees Celsius for 8 hours. Following the completion of the polymerization cycle, the flask was cooled slowly to room temperature. The retrieved acrylic samples from the dental flask were then subjected to finishing and polishing procedures.

Bulk reduction – tungsten carbide burs mounted on a laboratory lathe. Final contouring – acrylic trimmers and sandpapers (320, 400, 600 grits) Final polishing – pumice applied to rag wheel. The polished samples were segregated and stored in labeled boxes containing water. Total of eighty PMMA Heat Cure Denture Base Resin samples was fabricated and divided into two main categories: GROUP I: PMMA Heat Cure Denture Base Resin samples (n=40), GROUP II: PMMA Heat Cure Denture Base Resin samples incorporated with 8% EDTA (n=40). Samples in each group were further subdivided according to the testing parameters as follows:

Disc-shaped samples of size 10×1 mm were used for qualitative analysis of the antifungal property: (n=20) (Fig. 1A, Group I: n=10, GROUP II: n=1.

Figure 1. (A) Finished acrylic samples for qualitative analysis of antifungal property. (B) Finished acrylic samples for quantitative analysis of antifungal property.

Disc-shaped samples of size 10×1 mm were used for quantitative analysis of the antifungal property: (n=20), (Fig. 1B, Group I: n=10, Group II: n 10.

Strip-shaped samples of size 64×10×3.3 mm were used for testing the flexural strength. (Fig. 2, Group I: n=10, Group II: n=10.

Figure 2. Finished acrylic samples for evaluating flexural strength (64×10×3.3 mm).

Disc-shaped samples of size 50×0.5±0.05 mm were used for testing the color stability, (Fig. 3, Group I: n=10, Group II: n=10. The GROUP I and GROUP II samples for testing the antifungal property were immersed in a water bath at 37°C for 17 hours to reduce the residual monomer content. The samples for testing the flexural strength and color stability were immersed in the water bath at 37°C for 1 week to simulate the invivo environment.

Figure 3. Finished acrylic samples for evaluating Colour stability (50×0.5 mm).

1. Evaluation of antifungal property

The samples were sterilized in a UV chamber for 2 hours before incubation. Preparation of culture medium Sabouraud Dextrose Agar (SDA) medium preparation:

Sabouraud Dextrose Agar (SDA) medium is a growth medium commonly used for yeast cultivation. The medium was prepared by weighing 30 grams of SDA and 20 grams of Agar-agar by dissolving it in 1 liter of distilled water. The medium was boiled to completely dissolve it and the solution was then mixed and sterilized by autoclaving at 15 lbs pressure for 15 minutes at 121°C. The medium was then poured into the Petri plates.

Mueller-Hinton Agar medium is a microbiological growth medium commonly used for Antibiotic susceptibility testing. The medium was prepared by weighing 21 grams of MHA and 20 grams of Agar-agar by dissolving it in 1 liter of distilled water. The medium was boiled to completely dissolve it. The solution was then mixed and sterilized by autoclaving at 15 lbs pressure for 15 minutes at 121°C. The medium was then poured into the Petri plates. Media for cultural characteristics were prepared in the laboratory following the manufacturer's instructions. The specimens were cultured on Sabouraud Dextrose Agar (SDA) medium and Mueller-Hinton Agar (MHA) medium and the colony characteristics of the organisms were observed after overnight incubation at 37°C in the incubator. A single yeast colony of pure cultured Candida albicans (IFM40009) was inoculated into 10 ml of Sabouraud Dextrose broth for initial suspension. 100 µl of this initial suspension was serially diluted up to 10−5 in 900 µl saline. The suspensions 10−2, 10−3, 10−4 were plated by spread plate method onto SDA medium and incubated at 37°C overnight for Colony Forming Unit (CFU) enumeration. The acrylic discs were placed on Mueller-Hinton Agar plates, which were seeded with 1×106 CFU/ml. After plates were incubated for 24 hours at 37°C, the inhibition zone for candida growth was determined visually by measuring the diameter in mm using an antibiotic zone scale. Each acrylic disc was inoculated with 1,000 µl of Candida albicans suspension in Sabouraud Dextrose broth and was incubated with agitation at 25°C. After 24 hours of incubation, the acrylic discs were transferred into new sterile glass tubes containing saline and it was vortexed vigorously after which, 100 µl of suspension was serially diluted up to 10−5 in 900 µl saline. The diluted suspension (10−2, 10−3, 10−4) (Fig. 4, 5) was plated onto Mueller-Hinton agar plates by spread plate technique. All the plates were incubated for 24 hours at 37°C before reading. A colony counter was used to count the Colony Forming Units. The AntiCandidal effect was investigated by determining the reduction in colony counts against specimen growth control. Results were expressed as log CFU/ml for each specimen.

Figure 4. Disc Diffusion Method showing zone of inhibition.
Figure 5. Quantitative analysis of antifungal property-serial dilution.

CFU/ml=Total colony counted / Dilution×Volume

2. Evaluation of flexural strength (Fig. 6)

The samples were subjected to the Three-Point Bend test in Universal Testing Machine at a crosshead speed of 5 mm/min and the values were recorded using computer software. The load was delivered perpendicular to the center of the specimen until a fracture occurred. Flexural strength was calculated using the equation:

Figure 6. Sample in the Universal Testing Machine for flexural strength assessment.

FS=3FL / 2bd2, Where, FS - flexural strength in (MPa), F - load or force at fracture in (N), d-thickness, b-width, L - span length of specimen between two supports (50 mm).

3. Measurement of colour coordinates (Fig. 7)

CM-3600d Spectrophotometer was used to measure the Commission International function at 10°. The samples were dried and held against the aperture using the sample holder. The CIE system of the color specification provides a common means of analyzing and presenting color measurement data. To identify a particular object’s color, the spectral distribution of light reflected from the object should be known and that spectral reflectance must be averaged by three weighing functions called the color matching functions and they characterize the color matching properties of an average observer with normal color vision, known as the 1931 CIE standard observer. They are weighted by the relative spectral power distribution of CIE standard Illuminants. The resultants Tristimulus X, Y, and Z are the standard response of the eye to the red, green, and blue stimuli from the object.

Figure 7. Sample in the spectrophotometer.

Knowing the tristimulus values for a particular specimen is beneficial in terms of labeling its color. In discussing the color difference between objects, the CIELAB system can be employed. The magnitude and direction of the shift of the difference between two color stimuli can be identified. L* defines lightness, a*denotes red/green value, b*denotes yellow/blue value. The colors of each opponent pair are indicated by the positive and negative values of a* and b*. Calculation of (CIELAB) L, a, and b: From the tristimulus values the CIE L *, a*, and b* values were calculated by using CIE 1976 CIELAB equation. The L*, a* and b* values are derived from the tristimulus values X, Y, and Z as follows:

L*=116 (Y/Yn)1/3-16 a*=500[(X-Xn)1/3-(Y/Yn)1/3] b*= 200[(Y-Yn)1/3-(Z/Zn)1/3], Where, Xn, Yn, and Zn are the tristimulus values of the illuminant. For D 65 illumination at 2° observers. Along the*, axis color measurement movement in the +a direction depicts a shift toward red, and along the b* axis, +b movement represents a shift toward yellow. The center L* axis shows L=0 (black or total absorption) at the bottom. At the center of this plane, it is neutral or grey. These formulas, as well as those for determining and perceiving the color difference between the two objects (∆E) as follows:

ΔE*ab=[(ΔL*) 2+(Δa*) 2+(Δb*) 2] 1/2

∆L difference in lightness, ∆a difference in a coordinates, ∆b difference in b coordinate,

∆E –total color difference.

The expressions for these color differences are ΔL* Δa* Δb* (Δ symbolizes “delta,” which indicates difference). A CM-3600d spectrophotometer was used to analyze the Colour difference. The system was connected to the computer. The target mask was selected based on the specimen and application. The system was calibrated with a white calibration plate and zero calibration plate. The specimen was held against the aperture with the help of a sample holder and the L a b values were displayed in the system. The color difference was measured by the following formula:

ΔE=[(ΔL) 2+(Δa) 2+(Δb) 2]1/2

On a typical scale, the ΔE value will range from 0 to 100.


≤1.0 Not perceptible by human eyes

1∼2 Perceptible through close examination

2∼10 Perceptible at a glance

11∼49 Colors are more similar than the opposite

50∼100 Colors are the exact opposite

Data for qualitative and quantitative analysis of antifungal property of Group I and GROUP II samples with their mean value is given in Table 1. Data for evaluation of flexural strength of GROUP I and GROUP II and their mean value is given in Table 2, data for evaluation of color coordinates with mean L*, a*, b* values for GROUP I and GROUP II is given in Table 3 and 4. Qualitative and quantitative analysis of antifungal property of GROUP I and GROUP II samples performed using independent T-test as shown in Table 5 and 6. Comparative evaluation of flexural strength of GROUP I and GROUP II samples is given in Table 7. Comparative evaluation of color stability of GROUP I and GROUP II is given in Table 8.

Table 1 . Data for qualitative and quantitative evaluation of antifungal property of PMMA Heat Cure Denture Base Resin samples GROUP I and GROUP II against Candida albicans

Sample numberZone of inhibition-GROUP IZone of inhibition-GROUP IIColony forming unit (CFU)Colony forming unit (CFU)
10 mm14 mm116×1060
20 mm14 mm211×1060
30 mm14 mm148×1060
40 mm0 mm113×1060
50 mm16 mm87×106132×106
60 mm0 mm107×10692×106
70 mm0 mm96×1060
80 mm14 mm110×1060
90 mm20 mm84×106109×106
100 mm0 mm123×106117×106
Mean0 mm9.2 mm119.50×10645×106

Table 2 . Basic data for evaluation of Flexural strength of PMMA Heat Cure Denture Base Resin samples (GROUP I and II)

Sample numberFlexural strength (MPa) GROUP IFlexural strength (MPa) GROUP II

Table 3 . Basic data for evaluation of Colour coordinates of PMMA Heat Cure Denture Base Resin samples (GROUP II)

Sample NoLAB

Table 4 . Basic data evaluation of Colour coordinates of PMMA Heat Cure Denture Base Resin samples incorporated with EDTA (GROUP II)

Sample NoLAB

Table 5 . Comparative evaluation of Qualitative analysis of Antifungal property of GROUP I and GROUP II PMMA Heat Cure Denture Base Resin samples using Independent t-test

Disk diffusion testNMeanSDP-value
GROUP I10.0000.000000.002**

GROUP II109.20008.12130

If the P-value is 0.000 to 0.010 then denoted by **, it implies Significant at 1 level (Highly Significance).

Table 6 . Comparative evaluationof Quantitative analysis of Antifungalproperty of GROUP I and GROUP II PMMA Heat Cure Denture Base Resin samples using Independent t-test

Colony forming unitsNMeanStd. deviationP-value
GROUP I10119.5037.0680.003**

GROUP II1045.0058.886

If the P-value is 0.000 to 0.010 then denoted by **, it implies highly Significant.

Table 7 . Comparative evaluation Flexural strength of GROUP I and GROUP II PMMA Heat Cure Denture Base Resin samples using Independent t-test

Flexural strengthNMeanStd. deviationP-value
Group I10145.4336.541600.028*

Group II10112.5723.31785

If the P-value is 0.011 to 0.050 then denoted by * it implies Significant.

Table 8 . Comparative evaluation of color stability of GROUP I and GROUP II PMMA Heat Cure Denture base Resin samples

SAMPLEMean LMean aMean b
GROUP I41.045.92−1.07
GROUP II42.674.63−0.86
Δ E2.89

No zone of inhibition was formed for GROUP I and a mean zone of inhibition of 9.2 mm for GROUP II when evaluated with the Disc Diffusion method. The mean value of Colony Forming Units for PMMA Heat Cure Denture Base Resin samples is 119×106 mm. The mean value of Colony Forming Units for PMMA Heat Cure Denture Base Resin samples incorporated with EDTA is 45×106 mm. The mean value of flexural strength of PMMA Heat Cure Denture Base Resin samples is 145.43 MPa. The mean value of flexural strength of PMMA Heat Cure Denture Base Resin samples incorporated with EDTA is 112.57 MPa.

Foe Group I, The maximum value of L was 48.77 and the minimum 36.53, with a mean of 41.04. Maximum value of a was 8.29 and minimum 03.58 with a mean of 5.92. maximum value of b was 0.14 and minimum −02.16, with a mean of −1.07. For Group II, The maximum L value was 52.58 and minimum was 37.62, with a mean of 42.67. Maximum a value was 08.22 is and minimum value was 03.05, with a mean of 4.63. Maximum b value was 2.00 and the minimum was −02.01, with a mean of −0.86.


The main causative organism of Denture stomatitis is the Candida species. Studies have shown that Candida albicans is capable of adhering to mucous membranes and prosthesis16). Results from various studies3,17) have shown that no particular denture cleansing method is considered ideal over the other and also that combination of two or more methods is required in most conditions, which further makes denture hygiene maintenance more challenging. Scientific methods include incorporation of various organic and inorganic substances with antimicrobial properties or coating the denture base with materials creating hydrophobicity, and preventing adhesion of the microbial biofilm18). But these agents were found to considerably reduce the mechanical and optical properties of the PMMA resin.

The most frequent site of fracture in the upper denture is the midline15,19). Johnson and Mathew described the fact that on average, a denture flexes 5,00,000 times in a year. Smith had explained that recurring flexing from chewing creates denture fatigue. Resistance to flexure fatigue is a desirable property of denture base resin13,20). According to the ISO 1567:1999 standards for denture base polymers, flexural strength should not be less than 65 MPa21-23).

EDTA prevents the binding of Candida albicans to the proteins in a dose-dependent manner. EDTA reduces the growth of Candida albicans by removing calcium from the cell walls and causing collapses in the cell wall, and by inhibiting enzyme reactions. denture cleansers with EDTA and a mixture of enzymes (papain, lipase, amylase, trypsin) were effective in the removal of mucin, and heavy deposits of calculus from dentures17). In vitro studies have shown that EDTA can restrict Candida albicans biofilm formation through inhibition of its filamentation24,25). The results of the above study revealed that incorporation of EDTA in polymethyl methacrylate can enhance the antifungal property of PMMA Heat polymerized denture Base resin, with minimal reduction in flexural strength and color stability.

The result obtained is in line with the study conducted by Bilge et al.6) which showed that EDTA is more effective as an anticandidal agent than other antifungals like nystatin, ketoconazole, and chlorhexidine. Ejvind et al.17) showed that denture cleansers containing EDTA and a mixture of enzymes were fungicidal. Thus, it can be inferred that EDTA can be used to make PMMA resistant to Candida adherence and also that PMMA can be modified with antimicrobial agents to exhibit microbial resistance.

Though the flexural strength of both the groups is within the standards of ISO, Statistical analysis shows that this decrease is minimal and is significant (P-value 0.028*). results from this study are following the study conducted by Aysan1) which showed a decrease in flexural strength when PMMA was incorporated with an antimicrobial agent. However, a study by Mohamed and Reemet9) and Castro et al.26) showed that the incorporation of nanoparticles showed improvement in flexural strength. Srivastava25) and Neven15) explained changes in Flexural properties following a change in the percentage of antimicrobials added and type of polymer. Thus it can be concluded that incorporation of antimicrobial agents into PMMA may either enhance or deteriorate the mechanical properties, while EDTA evaluated in this study altered the flexural strength significantly.

The color stability of Polymethyl methacrylate Heat Cure Denture Base Resin (GROUP I) was evaluated using a Spectrophotometer and compared with that of Polymethyl methacrylate incorporated with EDTA (GROUP II). Thus ΔEvalue obtained was 2.89 which shows that the color difference is “perceptible at a glance. a study conducted by Hawraa27) to study the effect of Ti-O nanofillers showed a similar effect when the samples incorporated with Ti-O nanofillers showed increased opacity due to the presence of TiO2 within the matrix, which absorbs more light than the polymer matrix. ADA and ISO standards for evaluation of color stability of PMMA Heat Cure Denture Base Resin samples recommend that the resin shall not show, more than a slight color change, indicating that ΔE value ≥2.25 is not clinically acceptable when tested according to the standards. Hence the color change exhibited by the PMMA with the addition of EDTA requires additional investigation with a greater sample size.

The results obtained from the present in vitro study revealed that, though EDTA, 8% by weight, when incorporated in Polymethyl methacrylate heat polymerized Denture Base Resin enabled antifungal property, effects on mechanical and optical properties need further evaluation. Thus the null hypothesis of the present study is invalidated. This material could be used for making implant overdentures due to improved properties obtained, which could lead to the enhanced clinical performance of these overdentures especially reduced antifungal activity around attachments.

The present study had some limitations. The proportion of EDTA added to PMMA polymer is an important parameter that will decide the effect of EDTA on the Heat Cure Denture Base Resin. Exact reaction of EDTA with Polymethyl methacrylate was not analyzed.


The addition of EDTA into PMMA Heat Cure Denture Base Resin produced a significant antifungal effect by reducing the Candidal adherence to the PMMA samples. The presence of EDTA in the PMMA polymer has shown both flexural strength and color property reduction in a minimal manner. This improved PMMA resin can be used in clinical situations for implant overdentures, in addition to conventional use.

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