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 Table of Contents  
Year : 2019  |  Volume : 11  |  Issue : 3  |  Page : 118-121

Effect of chitosan on antibacterial activity of gypsum-based biomaterial compared to two dental liners

1 Conservative Dentistry Unit, School of Dental Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
2 School of Dental Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
3 Biomaterials and Prosthodontics Unit, School of Dental Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
4 School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia

Date of Web Publication25-Jun-2019

Correspondence Address:
Dr. Asma Abdullah Nurul
School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan
Dr. Hasan Subhi
Conservative Dentistry Unit, School of Dental Sciences, Universiti Sains Malaysia, Health campus, 16150 Kubang Kerian, Kelantan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jioh.jioh_25_19

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Aim: The aim of this in vitro study was to evaluate the antibacterial activity of gypsum-based biomaterial with different concentrations of chitosan (GP/CT) and to compare with the commercial dental materials, Dycal and glass ionomer cement (GIC) against the Streptococcus mutans. Materials and Methods: GP/CT was prepared with different concentrations of CT to GP of 0%, 10%, 20%, 40%, and 80% (w/w). The antibacterial study was carried out using agar diffusion method on Mueller-Hinton agar (MHA) against S. mutans. Uniform wells were prepared at equidistant points in the base layer of MHA. The wells were filled with the materials immediately after manipulation and preincubated for 2 h and then incubated. The means of inhibition zones were measured at 48 and 72 h. Results: The results revealed that higher means of inhibition zone were observed in a dose-dependent manner with higher concentrations of CT. GP/80%CT showed more effective antibacterial effect than GP/20%CT and GP/10%CT with significant degree and also with GP/40%CT with no significant result (P < 0.05), while GP/0%CT showed no inhibition of bacterial growth. GP/80%CT showed no statistically significant difference with GIC and Dycal on day 3. In addition, no significant bacterial growth inhibition was observed between GP/40%CT and GIC. Conclusion: Gypsum-based biomaterial has potent antibacterial activity against S. mutans when CT added. Higher antibacterial activity of GP/CT biomaterial was observed in a dose-dependent manner with higher concentrations of CT. GP/80%CT exhibits the antibacterial effect similar to that of Dycal and GIC.

Keywords: Antibacterial activity, Chitosan, Gypsum, Streptococcus mutans

How to cite this article:
Subhi H, Hakimi I, Jie NT, Reza F, Husein A, Nurul AA. Effect of chitosan on antibacterial activity of gypsum-based biomaterial compared to two dental liners. J Int Oral Health 2019;11:118-21

How to cite this URL:
Subhi H, Hakimi I, Jie NT, Reza F, Husein A, Nurul AA. Effect of chitosan on antibacterial activity of gypsum-based biomaterial compared to two dental liners. J Int Oral Health [serial online] 2019 [cited 2022 Jan 26];11:118-21. Available from:

  Introduction Top

The main reason that contributes to the restoration failure in the dentistry is the existence of secondary caries.[1],[2] When fermentable carbohydrates are present, the plaque bacteria such as Streptococcus mutans invade and initiate a demineralization of tooth structure between the cavity wall and the restoration, leading to secondary caries formation.[1] The antibacterial activity of the restorative materials inactivates the remaining bacteria and microleakage, preventing secondary caries formation, and provides a long-lasting restoration. Recently, the interests in enhancing the antibacterial properties of restorative materials are focused on incorporation of antibacterial agents in the composite.[3],[4],[5],[6]

Chitosan (CT), a deacetylated derivative of chitin, is a naturally acquired polysaccharide obtained mainly from shrimp shells and crabs.[7] CT is nontoxic, biocompatible, and has antibacterial activity.[8],[9] CT is highly desirable in the dental materials because of its antibacterial properties and biocompatibility. CT has been studied in many aspects of dentistry such as antibacterial agent in dental materials,[10],[11] in dental tissue engineering,[12],[13],[14] and as a component in toothpaste to reduce plaque formation and abrasion.[15],[16],[17]

Gypsum “calcium sulfate” (GP) was used as a resorbable base material in this study. GP is safe,[18] well tolerated, and has inherent osteoconductive properties.[19] It can be employed to deliver pharmacologic agents, antibiotics, and growth factors.[20],[21] Thus, CT was incorporated into GP to synthesize a biocompatible antibacterial material for dental pulp lining.

The aim of the present study was to evaluate the effect of CT concentrations on the antibacterial activity of gypsum-based biomaterial against S. mutans by comparing with the commercial restorative materials, Dycal and GIC.

  Materials and Methods Top

This experimental biological study was performed using agar diffusion method against the strain of S. mutans in three technical replicates and three independent biological experiments for each material group. All the culture procedures were conducted under sterile conditions.

The details of the materials used are listed in [Table 1]. Water-soluble CT chloride “referred to as CT in this study” was used. Calcium sulfate dihydrate was heated at 200°C for 5 h to convert it to hemihydrate form. GP/CT biomaterial was prepared with five different concentrations of CT to GP of 0%, 10%, 20%, 40%, and 80% (w/w). 250 mg of GP and CT powder was mixed with distilled water at a volume of 190, 200, 300, 400, and 600 μL, respectively, to obtain a moldable paste that sets in few minutes. Dycal and GIC were mixed according to the manufacturer's instructions.
Table 1: Materials used in the study

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Agar diffusion method

[Figure 1] shows the flowchart of the test procedures. The antibacterial activity of the test materials was evaluated using Mueller-Hinton agar (MHA). S. mutans (ATCC 35668) was cultured on blood agar at 37°C for 48 h in anaerobic environment; the bacteria were then harvested and suspended in Mueller-Hinton broth for overnight. The concentration was diluted to obtain a 0.5 McFarland turbidity standard (corresponding to 1.5 × 108 CFU/ml). Bacterial suspension was poured onto the MHA surface and the excess was extracted. Uniform wells (5 mm in diameter, 5 mm in height) were made at equidistant points in MHA with a sterile copper coil. The test materials were mixed on a sterile glass slab by a sterile spatula and placed immediately in the wells using a sterile amalgam carrier and condensed gently. Plates with and without inoculums incubated under the identical conditions and for the same period served as positive and negative controls.
Figure 1: Flowchart of the study experiment

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The test materials were prediffused for 2 h at room temperature. Then, the plates were incubated in anaerobic condition at 37°C. The bacterial inhibition zones were evaluated after 48 and 72 h with a precision rule to the closest 0.5 mm. The results were represented as the mean and standard deviation.

Statistical analysis

Statistical analyses were performed using SPSS (Version 22.0; SPSS, Chicago, IL, USA). Statistical significance was evaluated by one-way analysis of variance followed by Dunnett's T3 post hoc test for multiple comparisons of each pair if there was a significant difference; P < 0.05 was considered statistically significant.

  Results Top

The antibacterial activity of GP/CT, GIC, and Dycal against S. mutans is shown in [Figure 2] and [Figure 3]. Bacterial growth was observed in positive control, whereas no bacterial growth was observed in negative control. The test material showed antibacterial activity against S. mutans with different intensities when CT was added. Higher means of inhibition zones were observed in a dose-dependent manner with higher concentrations of CT.
Figure 2: Antibacterial activity of GP/CT biomaterial, GIC, and Dycal. Values are expressed as mean ± standard deviation of diameter of inhibition zones in mm (n = 9). *P < 0.05 against GP/0%CT, #P < 0.05 against GP/10%CT, P < 0.05 against GP/20%CT, ¥P < 0.05 against GP/40%CT, P < 0.05 against GP/80%CT. GP/CT: Gypsum-based biomaterial; GIC: Glass ionomer cement

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Figure 3: Inhibition zones promoted by GP/CT biomaterial, GIC, and Dycal against Streptococcus mutans. GP/CT: Gypsum-based biomaterial; GIC: Glass ionomer cement

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The results revealed that GP/80%CT biomaterial demonstrated no statistical significant with GIC and Dycal on day 3. However, no significant inhibition in the bacterial growth could be observed between GP/40%CT and GIC. In addition, GP/80%CT showed more effective antibacterial effect than GP/20%CT and GP/10%CT with significant inhibition (P < 0.05). However, no statistically significant was found between GP/40%CT and GP/20%CT, but GP/40%CT showed significant difference with GP/10%CT (P < 0.05). On the other hand, GP/0%CT showed no inhibition of bacterial growth.

  Discussion Top

In the present study, we investigated the antibacterial activity of gypsum-based biomaterial with different concentrations of CT and compared that with Dycal and GIC. Although calcium sulfate has no antimicrobial activity, it is not a good material for the growth of some microbes, and it can be used as a scaffold to deliver therapeutic drugs and antibacterial agents;[20] thus, it was applied as a resorbable scaffold to release the active antibacterial component of CT.[21] S. mutans frequently implicated in dental caries and pulpal inflammation[22] and has the main role in the initial lesion and pulpal pathology.[23] The agar diffusion technique, a widely used method for the assessment of antimicrobial activity,[24] was employed in this study. Many researchers have used this technique in antibacterial researches.[25],[26] However, some factors may interfere with the inhibition zone formation around the materials including strains of the bacteria, diffusion capacity of the antimicrobial agents, cellular density, agar medium, and anaerobic atmosphere.[27]

The present study revealed that GP/CT biomaterial has potent antibacterial activity with a larger zone of bacterial growth inhibition in high concentration of CT, while pure gypsum was incapable to inhibit the growth of S. mutans.

The results showed that GP/80%CT had the highest inhibition zone among the experimental materials, but not to a significant degree with GP/40%CT. Both GP/80%CT and GP/40%CT showed antibacterial activity similar to that of GIC. However, Dycal showed higher antibacterial activity than GP/40%CT. The antibacterial efficacy of CT is related to the interaction between the amino groups of CT with a positive charge and the cell surfaces of the microbes with a negative charge. This interaction results in the leakage of proteinaceous and intracellular material due to the loss of the microbes' barrier function.[28] The low CT concentration biomaterials (GP/10%CT and GP/20%CT) had the lowest antibacterial activity with significantly lower mean of inhibition zone than GP/80%CT. Fei Liu et al.[29] stated that the antibacterial activity of CT in the medium increases when the CT concentration increases. As the bacterial inhibitory actions of CT depend on the amount of NH3+ and its strength is increased with higher NH3+ concentration, which is indicated by CT, the finding is comparable with the results obtained in our study. GP/0%CT did not show any activity against S. mutans, indicating that the antibacterial activity of the GP/CT biomaterial is related to the action of the CT.

Dental liners affect the bacterial growth by direct contact with the bacteria and diffusion into the dentinal tubules. In addition, the effect of dental liners on the bacterial growth originates from the antibacterial activity of the liner and/or the change of environmental conditions. Calcium hydroxide is an optimal medication for many clinical situations because of its antimicrobial action and stimulation of reparative dentin.[30] It was shown in our results that Dycal had a strong antibacterial effect, confirming the results of others.[31],[32] This antibacterial effect may be related to the release of highly oxidant and free radicals hydroxyl ions, which shows extreme reactivity with several biomolecules.[33] GIC is well known in its advantage to inhibit dental caries; the antibacterial activity of GIC is attributed to the low pH and fluoride release.[34] The present study demonstrated that GP/CT biomaterial has potent antibacterial activity comparable with Dycal and GIC and the efficient of the bacterial inhibition varies significantly according to the concentration of CT.

The key strength of this study was the comparison of the synthesized materials with the commercial restorative materials – Dycal and GIC – which possess excellent antibacterial properties. However, this research was limited to one cariogenic bacterium and the evaluation of other cariogenic bacteria would be a fruitful area. This study is a brief attempt to evaluate the role of CT on gypsum-based biomaterial. Its effects against S. mutans are not a novelty, but the role of CT does provide a new treatment approach, which can be further researched.

  Conclusion Top

Gypsum-based biomaterial has potent antibacterial activity against S. mutans, while pure GP did not show inhibition of bacterial growth. Higher antibacterial activity of GP/CT biomaterial was observed in a dose-dependent manner with higher concentrations of CT. Further experimental investigations are needed to access the antibacterial activity of GP/CT biomaterial against various bacteria involved in dental caries.


We would like to thank the staffs of Craniofacial Science Laboratory, School of Dental Sciences, Universiti Sains Malaysia, for their technical support.

Financial support and sponsorship

This study was supported by short-term grants, No. 304/PPSG/61313006 and No. 304/PPSG/61312099 of Universiti Sains Malaysia.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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