**4. Anticariogenic effects of chitosan**

Experiments were performed in the Microbiology Laboratory-Nucleus of Research in Environmental Sciences- UNICAP (Recife, PE, Brazil) regarding the safety concentration for the dilution of fungi chitosan in acetic acid. The mechanisms of chitosan from crabs and fungi to inhibit the tooth colonization by *S. mutans, S. sanguis, S. mitis* and *S. oralis* were evaluated through the adherence test of chitosan to dental and bacteria surface, results showed in Figure 2. Chitosan from crabs and fungi, in all concentration tested, decreased the adsorption of *Streptococcus* strains to dental enamel, reduced the bacteria cell wall hydrophobicity and decreased the glucan production by bacteria. However, chitosan from fungi was more efficient than chitosan from crabs for the tree parameters studied.

Microbiological Chitosan: Potential Application as Anticariogenic Agent 237

inhibiting effects of chitosan may depend, at least, partially, on alterations of bacterial cell

In figure 2C is observed a decreasing of extracellular glucan production by bacteria strain in presence of sucrose, with increasing chitosan concentration. The ionic interaction between the cation, according to [61,62], from chitosan (amine group) and anionics parts of bacteria cell wall (phospholipids and carboxylic acids) can form a membrane polymer, which prevents nutrients from entering the cell. Since chitosan could adsorb the electronegative substance, this polymer can promote cell flocculate, and disturbs the physiological activities of the bacteria and kill them. Authors [63] reported that chitosan interact with the electronegative bacterial cell surface resulting in displacement of Ca++ from anionic

membrane sites, resulting in a changing the electric potential of the bacteria surface.

0.25

1

2 4 6 8 10 12 14 16 18 20 Adherence of bacteria on tooth (%) chitosan from fungi

0.5

0.05

0.05

0.025

0.05

0.025

0.25

0.25

0.5

0.25

0.02

0.025

*S. mutans S. mitis S. oralis S. sanguis* 

0.5

1

5

0.5

1

2 3 4

0.5

0.25 0.5 <sup>1</sup>

1

0.5

0.25 0.5 1

1

Hidrophobicity (%)

Chitosan from crabs

2

1

*S. mutans S. mitis S. oralis S. sanguis*

<sup>4</sup> <sup>5</sup>

5

2 3

Adherence of bacteria on tooth (%)

chitosan from crabs

2 <sup>3</sup> <sup>4</sup> <sup>5</sup>

3 4

A

0.25

0.05

0.25

B

5 15 25 35 45 55 65 75 85 95 Hidrophobicity (%) Chitosan from fungi

surface hydrophobicity expression.

Figure 2A shows the decrease of bacteria adsorption to dental enamel in the presence of chitosan from crabs and fungi, in all concentrations studied. Chitosan demonstrated best performance at the concentration of 2mg/mL for *S. mutans* and of 3mg/mL for *S. sanguis, S. mitis* and S*. oralis*. These results are in agreement with the one obtained by [8], which studied the effect of chitosan from crabs of low molecular weight in the adsorption of *S. mutans, S. sanguis* and *S. oralis* to the commercial hydroxyapatite.

Researchers [9, 10] investigated, "in vivo", the activity of a chitosan mouthrinse, respectively of 1% and 0.5%, and verified significant reduction of dental plaque formation. The authors reported that chitosan might be altering of the electrostatic interaction between the bacterial cell surface in saliva and tooth pellicle surface. The electrostatic interaction is usually repulsive due to the fact that inature both bacteria and the pellicle surface are predominantly negatively charged. The chitosan chains attach themselves to the negatively charged bacterial cell surface by means of their positively charged groups. If these chains are of a sufficient length to bind more than one cell, bridges are formed between bacterial cells. As soon as the bridging becomes effectives flocs are formed, and the bacteria cannot colonize the tooth surface.

In literature [8-10] is reported that aggregating oral bacteria may reduce their adherence to tooth surface. The polycationic nature of chitosan might reduce the initial bacterial adherence onto the teeth surfaces, at least in part, by generating bacterial aggregation. There have also been suggestions that bacterial aggregates are removed more easily from the oral cavity than individual bacterial cells.

To verified the influence of chitosan, sublethal concentration, to modification in bacterial cell surface, it was evaluated the affinity of chitosan to xylene of bacteria grown in the presence or absence of chitosan sub-MICs, through hydrophobicity tests (Figure 2B), and for sucrose catabolization, through extracellular glucan production by bacteria strains (Figure 2C). The results in figure 2B indicates that increasing concentration of chitosan in bacterial suspension caused a successive decrease of the bacteria cell wall hydrophobicity, being more evident for the *S. mutans* strain. These results are supported by findings of [8,9]. These authors suggested that chitosan induced a successive decrease in cell hydrophobicity, and that surface hydrophobicity is related to adherence ability of bacteria. Therefore, the inhibiting effects of chitosan may depend, at least, partially, on alterations of bacterial cell surface hydrophobicity expression.

236 Practical Applications in Biomedical Engineering

colonize the tooth surface.

cavity than individual bacterial cells.

**4. Anticariogenic effects of chitosan** 

Experiments were performed in the Microbiology Laboratory-Nucleus of Research in Environmental Sciences- UNICAP (Recife, PE, Brazil) regarding the safety concentration for the dilution of fungi chitosan in acetic acid. The mechanisms of chitosan from crabs and fungi to inhibit the tooth colonization by *S. mutans, S. sanguis, S. mitis* and *S. oralis* were evaluated through the adherence test of chitosan to dental and bacteria surface, results showed in Figure 2. Chitosan from crabs and fungi, in all concentration tested, decreased the adsorption of *Streptococcus* strains to dental enamel, reduced the bacteria cell wall hydrophobicity and decreased the glucan production by bacteria. However, chitosan from

Figure 2A shows the decrease of bacteria adsorption to dental enamel in the presence of chitosan from crabs and fungi, in all concentrations studied. Chitosan demonstrated best performance at the concentration of 2mg/mL for *S. mutans* and of 3mg/mL for *S. sanguis, S. mitis* and S*. oralis*. These results are in agreement with the one obtained by [8], which studied the effect of chitosan from crabs of low molecular weight in the adsorption of *S.* 

Researchers [9, 10] investigated, "in vivo", the activity of a chitosan mouthrinse, respectively of 1% and 0.5%, and verified significant reduction of dental plaque formation. The authors reported that chitosan might be altering of the electrostatic interaction between the bacterial cell surface in saliva and tooth pellicle surface. The electrostatic interaction is usually repulsive due to the fact that inature both bacteria and the pellicle surface are predominantly negatively charged. The chitosan chains attach themselves to the negatively charged bacterial cell surface by means of their positively charged groups. If these chains are of a sufficient length to bind more than one cell, bridges are formed between bacterial cells. As soon as the bridging becomes effectives flocs are formed, and the bacteria cannot

In literature [8-10] is reported that aggregating oral bacteria may reduce their adherence to tooth surface. The polycationic nature of chitosan might reduce the initial bacterial adherence onto the teeth surfaces, at least in part, by generating bacterial aggregation. There have also been suggestions that bacterial aggregates are removed more easily from the oral

To verified the influence of chitosan, sublethal concentration, to modification in bacterial cell surface, it was evaluated the affinity of chitosan to xylene of bacteria grown in the presence or absence of chitosan sub-MICs, through hydrophobicity tests (Figure 2B), and for sucrose catabolization, through extracellular glucan production by bacteria strains (Figure 2C). The results in figure 2B indicates that increasing concentration of chitosan in bacterial suspension caused a successive decrease of the bacteria cell wall hydrophobicity, being more evident for the *S. mutans* strain. These results are supported by findings of [8,9]. These authors suggested that chitosan induced a successive decrease in cell hydrophobicity, and that surface hydrophobicity is related to adherence ability of bacteria. Therefore, the

fungi was more efficient than chitosan from crabs for the tree parameters studied.

*mutans, S. sanguis* and *S. oralis* to the commercial hydroxyapatite.

In figure 2C is observed a decreasing of extracellular glucan production by bacteria strain in presence of sucrose, with increasing chitosan concentration. The ionic interaction between the cation, according to [61,62], from chitosan (amine group) and anionics parts of bacteria cell wall (phospholipids and carboxylic acids) can form a membrane polymer, which prevents nutrients from entering the cell. Since chitosan could adsorb the electronegative substance, this polymer can promote cell flocculate, and disturbs the physiological activities of the bacteria and kill them. Authors [63] reported that chitosan interact with the electronegative bacterial cell surface resulting in displacement of Ca++ from anionic membrane sites, resulting in a changing the electric potential of the bacteria surface.

B

Microbiological Chitosan: Potential Application as Anticariogenic Agent 239

shown antibacterial action for bacteria belonging to the group *Streptococcus*, inhibits the growth and adherence of cariogenic bacteria and the desmineralization process of dental enamel in vitro, and stimulates salivation, *in vivo*. The results confirm a high biotechnological potential for chitosan from both fungi and crab sources as a cariostatic and anticariogenic agent, suggesting their application as dentistry biomaterial for prevention

*Department of Physiology and Pathology, Federal University of Paraíba, Paraíba-PB, Brazil* 

*Post-graduate in Materials Science, Federal University of Pernambuco, Recife-PE, Brazil* 

*Department of Pharmaceutical Science, Federal University of Paraíba, Paraíba-PB, Brazil* 

*Nucleus of Research in Environmental Sciences, Catholic University of Pernambuco, Recife-PE,* 

The authors are gratefully to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco

[1] Tahmourespour A, Kermanshahi RK, Saleh R, Pero NG. Biofilm formation potential of oral streptococci in related to some carbohydrate substrates. African Journal of

[2] Stamford-Arnaud TM, Barros Neto B, Diniz FB. Chitosan effect on dental enamel deremineralization: an in vitro evaluation. Journal of Dentistry. 2010; 38 (11) 848-852. [3] Keegan GM, Smart JD, Ingram MJ, Barnes LM, Burnett GR, Rees GD. Journal of

[4] Fitzgerald RJ, Keyes PH. Demonstration of the etiologic role of streptococci in experimental caries in the hamster. Journal of the American Dental Association. 1960;

[5] Carvalho MMSG, Stamford TCM, Santos EP, Tenorio P, Sampaio F. Chitosan as n oral antimicrobial agent. In: Mendez A (ed). Science against microbial pathogens:

Horacinna Maria de Medeiros Cavalcante and Rui Oliveira Macedo

Microbiology Research. 2010; 4 (11) 1051-1056.

Dentistry. 2012; 40, 229-240.

and therapeutic of dental carie.

Thayza Christina Montenegro Stamford

Thatiana Montenegro Stamford-Arnaud

Galba Maria de Campos-Takaki\*

**Acknowledgement** 

**Author details** 

*Brazil* 

(FACEPE).

**7. References** 

61, 9-19.

Corresponding Author

 \*

**Figure 2.** Diagram of stratification dispersion of chitosan from fungi and crabs activity: inhibition of bacteria adsorption on dental enamel (A); hydrophobicity of bacteria surface (B) and glucan production by bacteria (C).

Experiments were conducted in the department of chemistry's fundamental UFPE verified the penetration depth of chitosan in teeth enamel for two specimens by Optical Coherence Tomography (OCT) images. Chitosan was applied only to half of the teeth surface. The image in the left was obtained for a chitosan concentration of 1.25mg/ml. it is clearly seen the penetration of chitosan as indicated by the brighter area (caused by light scattering) in the figure, only in the region where chitosan may act as a mechanical barrier for the acid penetration in the enamel, which would explain its effect in the demineralization inhibition [2].

Sources: [2]

**Figure 3.** Optical Coherence Tomography (OCT) images of specimens treated with chitosan at 1.25mg/mL (left) and 5.0mg/mL (right)

#### **6. Conclusion**

The use of chitosan in different formulations, such as toothpaste (Chitodent®), mouthwash solution and chewing gum, is mentioned in literature [8-10]. In all forms the chitosan has shown antibacterial action for bacteria belonging to the group *Streptococcus*, inhibits the growth and adherence of cariogenic bacteria and the desmineralization process of dental enamel in vitro, and stimulates salivation, *in vivo*. The results confirm a high biotechnological potential for chitosan from both fungi and crab sources as a cariostatic and anticariogenic agent, suggesting their application as dentistry biomaterial for prevention and therapeutic of dental carie.
