Scientific background

A scientific report on chitosan as an ingredient
of the toothpaste Chitodent

Dr. Simone Geyer
University of Oldenburg
Faculty of Mathematics and Science
Carl von Ossietzky Str. 9-11
D 26111 Oldenburg

on behalf of

Helmuth Focken
Sundermann Straße 18
D 26835 Hesel


The toothpaste Chitodent consists of chitosan. Chitosan is a biopolymer derived from chitin by deacetylation. The molecule size varies widely and is characterized by either the average degree of polymerization or the molecular weight of the polymer. Depending on chain lenght, chitosan derivates are divided into two groups - low-molecular weight and high-molecular weight (MW 10,000 – 2,000,000 Da). The degree of deacetylation characterizes the amount of reactive amino groups. In addition, the environmental conditions such as pH, temperature and ionic strength strongly influence the biological activity of chitosan and its physicochemical properties, e.g. viscosity.

Chitosan is widly used in dental medicine due to it’s broad spectrum of biological activities:

The following study of the current literature summarizes the state of knowledge of chitosan application and its properties.

Attributes of chitosan and chitin

Chitosan is a biopolymer, which is used in different fields of the industry, medicine and cosmetic industry. Chitosan is obtained from shrimp, crab shell and squid by deacetylation resulting in a molecule having reactive amino groups (Schanzenbach, 2000). Chitosan is a linear 1,4 beta-linked polysaccharide comprising copolymers of glucosamine and N-acetylglucosamine (Illum, 1998). Chitosan derivates are defined by the following characteristics:

Chitosan and chitin are of commercial interest due to their biocompatibility and biodegradability. In addition, chitosan is nontoxic, inexpensive (Illum, 1998), and can easily be modified (Muzzarelli, 1988; Rabea et al., 2003). Chitosan is known for its antimicrobial activity, antitumor activity, haemostatic activity and acceleration of wound healing process (Muzzarelli, 1988; Chandy & Sharma, 1990; Illum, 1998; Kurita, 1998; Hirano, 1999). The use of chitosan is limited because of its insolubility in water, high viscosity, and tendency to coagulate with proteins at high pH (Rabea et al., 2003).

The chitosan used in the toothpaste Chitodent was made by deacetylation using chitin taken from squid. It is pure in a high degree, free of proteins and minerals; characterized by a high-molecular weight (MW: 972,073 Da) and a high degree of deacetylation (DDA: > 95 %).

Antimicrobial activity of chitosan

The antimicrobial activity of chitosan has been observed with a wide variety of microorganisms including fungi, viruses and some bacteria (Hirano & Nagano, 1989; Chirkov, 2002; Rabea et al., 2003). It was found that the antimicrobial activity of chitosan is influenced by intrinsic factors as well as environmental conditions:

Most of the information on the antimicrobial activity of chitosan derives from experiments with the gram-negative bacterium Escherichia coli, which is often used as a model organism.

Plaque, caries and periodontitis caused by human pathogenic bacteria

The antibacterial activity of different chitosan derivates depends on the analysed bacterial strains (Chen et al., 1998; Rabea et al., 2003; Chung et al., 2004). Due the fact that chitosan demonstrates film forming ability and gelation characteristics, it is used in the dental industry and cosmetic industry. The antibacterial activity of chitosan derivates is described for typical pathogens causing caries and plaque. Some Streptococcus members (gram-positive bacteria) are human pathogen and responsible for plaque and caries formation.
The antibacterial activity of differnt low-molecular weight chitosan derivates was shown on Streptococcus mutans (Fujiwara et al., 2004). Dental caries is the most common oral disease in humans. Dental caries is caused by the demineralisation of dental hard tissues, enamel and dentine, which occurs through fermentation of indigenous bacteria, like Streptococcus mutans. When saliva-coated or –uncoated hydroxyapatite2 breads were treated with chitosan, a reduction of bacterial growth was observed (Tarsi et al., 1997). In addition, Fujiwara et al. (2004) described the inhibition of S. mutans at pH 6, using different chitosan derivates (polymer, oligomer, and monomer). This result suggests that the antibacterial activity of chitosan derivates was independend of chitosan’s molecular weight and degree of polymerisation. However, Kurita (1998) showed the influence of the molecular weight of chitosan (10,000/70,000/220,000/426,000 Da) on the antibacterial activity of chitosan against different S. mutans strains. The low-molecular weight chitosan derivate (10,000 Da) reduced the viable cell count of S. mutans of 13 - 60 %. Whereas high-molecular weight chitosan derivates (70,000/220,000/426,000 Da) reduced the viable cell count to more than 90 %.
Analyzing the antibacterial activity of chitosan derivates using other genera of Streptococcus species, different results have been obtained (Tarsi et al., 1998; Decker et al., 2005). Chitosan derivates inhibites the growth of S. mutans, but did not inhibit the growth of other Streptococcus species (Tarsi et al., 1997, 1998). Only the chemical modification of chitosan derivates (N-carboxymethyl-chitosan and imidazolyl-chitosan) reduced the viable cell count of all investigated Streptococcus species. Decker et al. (2005) investigated the antibacterial activity of two chitosan derivates using planctonic S. sanguinis cells and S. sanguinis cells attached on enamel slides. The antibacterial activity of chitosan was influenced by both physicochemical properties of chitosan derivates as well as environmental conditions. Both chitosan derivates showed only minor antibacterial activity on planctonic cells. In contrast, one of the used derivate efficiently inhibited the growth of the attached cells. An increasing antibacterial activity of both chitosan derivates on planctonic as well as on attached cells was demonstrated after combined application of chitosan and chlorhexidin.

Periodontitis3 is caused by Porphyromonas gingivalis. P. gingivalis  has been considered as an aggressive periodontal pathogen due to of its high association with periodontal destruction in humans, and it is reported to prolong inflammation and progression of attachment loss. The impact of chitosan formulation (either as gel or film) against the periodontal pathogen P. gingivalis was investigated. Furthermore the viscosity, bioadhesive properties and antibacterial activity of different chitosan derivates (different molecular weight and degree of deacetylation) were investigated (Ikinci et al., 2002). Both, the chitosan gel and the chitosan film exerted bioadhesive properties. Chitosan is shown to have an antimicrobial activity against P. gingivalis, and this effect was even higher using high-molecular weight chitosan. However, the antibacterial activity which was achieved when the low concentrated high-molecular weight chitosan gel (1 %) was applied was similar to that of the high concentrated (3 %) low-molecular weight chitosan gel. Changes in the degree of deacetylation did not have any effect on antibacterial activity.

Conclusion: The antibacterial activity of different chitosan derivates was demonstrated for pathogens causing plaque, caries and periondontitis. It was shown that different factors influenced the antibacterial activity of chitosan. In every case, acidic pH seems to be optimal. The efficiency of antibacterial activity of low- and high-molecular weight chitosan derivates was also determined by using bacterial species. In addition, there are some hints that the efficiency of antibacterial activity could be regulated by chitosan concentration, which means that the antibacterial activity of a low concentrated high-weight chitosan could be nearly the same as that of a high concentrated low-weight chitosan derivate.

Chitosan stimulates wound healing

Chitosan and chitin in vivo have wound healing enhancing properties. Numerous reports describe the stimultory effects of chitosan on tissue reactions involved in wound healing. Chitosan has widely been used as an effective agens in various medical fields and dentistry in particular (Chandy & Sharma, 1990; Shigemasa et al., 1995; Kas, 1997; Illum, 1998; Hirano, 1999; Muzzarelli et al., 1999). Chitosan has many useful and advantageous biological properties in the application as a wound dressing:

The healing of wounds proceeds in three overlapping phases: inflammation, granulation tissue4 formation, and the matrix and remodeling phase. Based on the chitosan properties, described above, some authors describe a stimulation effect on wound healing process (Chandy & Sharma, 1990; Hirano, 1999; Kojima, 2004). Chitosan stimulates the fibroblastic cells to release chemotactic inflammatory cytokinesis (Mori, 1997; Muzzarelli et al., 1989). Histological findings indicated that chitosan induces the migration of polymorphonuclear leukocytes and macrophages in the investigated tissue at the early stage (Hidaka et al., 1999; Lu et al., 1999). At the final stage of wound healing process using chitosan, angiogenesis5, reorganisation of the extracellular matrix, and granulation tissue have been demonstrated. Furthermore, chitosan was able to establish a film, which is water insoluble but allows the penetration of oxygen (Shigemasa & Minami, 1995). The established chitosan based film is biodegrable.

The stimulation of wound healing after application of chitosan derivates was investigated using model organisms like rats. In vivo and in vitro, a positive effect on wound healing process was demonstrated using a chitosan monomer (MW: 215.6 Da) and different kinds of chitosan polymers (MW: 80,000/191,000/300,000 Da; DDA: 80/85 %) (Shigemasa & Minami, 1990; Kojima et al., 2004; Matsanuga et al., 2006). To obtain effective wound healing accelerator based on chitosan (MW: 200,000 Da; DDA: 90 %), a water soluble chitosan/heparin complex was prepared (Kweon et al., 2003). Kweon et al. (2003) used the wound healing properties of chitosan and the heparin properties to attrac or bind growth factor related to wound healing process. The high viscosity of this chitosan/heparin complex (7.24 and 7.32 Pa s) could enhance its direct usage. On the basis of this study, the results demonstrated that the wound healing in vivo was most effective by using a chitosan/heparin complex. In addition, the wound healing process using chitosan only, was even better than the wound healing process without chitosan (Kweon et al., 2003). Ishihara (2002) prepared photo-crosslinkable chitosan molecules, which can easily be applied to various kinds of wounds. The authors produced insoluble hydrogel by exposing it to short ultraviolet light irradiation. This chitosan gel (chitosan MW: 800,000 – 1,000,000 Da; DDA: 80 %) contained both lactobionic acid and p-azidobenzoic acid. Wound healing experiments using a mouse model have shown that the application of chitosan hydrogel on open wounds induced significant wound contraction and accelerated the wound closure and healing. After wound application combined with a short UV irradiation, which seemed to have no detectable side effects on the organism’s skin, an insoluble, non-toxic, and strongly adhesive wound closures was established. The photo-crosselinked gel was used for wounds of different model organisms such as pigs, rabbids, and rats. The additional UV irradiation stimulated the wound healing process effectivly.

Chitosan usage in dentistry

In the dental medicine, reserach is focused on gel development to allowe an easy application on open wounds in oral cavity. The clinical therapeutic approach to the problems of periodontitis has shown a variety of pathways. Biocompatible materials like chitosan and its modifications were used for the reduction of the periodontal pockets in the surgical interventions. Chitosan ascorbate, obtained by mixing chitosan with ascorbic acid and sodium ascorbate, was produced in gel, suitable for the treatment of periodontitis (Muzzarelli et al., 1989). Chitosan was progresively reabsorbed by the host, with a good clinical recovery, tested in 52 patients. In vivo, the tooth mobility and tooth pocket depths were significantly reduced. Bumgardner et al. (2003) used chitosan as bioactive coating to improve osseointegration of orthopaedic and cranifacial implant devices. Coating material was made from 91.2 % deacetylated chitosan (MW: 200,000 Da), which was chemical linked to titanium coupons. The bonded coatings exhibited minimal degradation within 8 weeks in cell cultures. They supported increased osteoblastic cell attachment and proliferation as compared to uncoated titanium controls. A relation between high grade of deacetylation and low degration of polymers was demonstrated.

Conclusion: Chitosan exhibits some properties, which make it attractive for usage in dental medicine in order to increase the wound healing process. Chitosan

Heavy metal binding properties of chitosan

Biosorption is recognized as an emerging technique for the detoxification of heavy metals. It is well-known that chitin and chitosan have the ability to selectively take up heavy metal ions (Kurita et al., 1979; Randall et al., 1979). The high amount of amino groups in this natural polymer results in novel binding properties of metal ions such as cadmium, copper, leed, uranyl, mercury and chromium (Eiden et al., 1980). The heavy metal uptake capacitiy of chitosan depends on the pH. The primary amino groups within the chitosan molecule are responsable for chemical modification to ligands and complex formations, resulting in new chitosan derivates, which are able to bind heavy metals selectivly, e.g. from waste water.

The adsorption of heavy metals (mercury chloride and copper chloride) by chitosan depends on the amount of reactive amino groups (Kurita et al., 1979). Chitosan derivate with higher amino group content (between 15 - 50 %) have a higher adsorption ablitiy compared to those having a lower content of amino groups. However, if the amino group content is higher than 50 % the adsorption properties of chitosan were no longer influenced by the degree of deacetylation6. Comparing the capacity of chitosan and chitin to adsorb heavy metal such as lead and chromium, chitosan has shown the most effective binding properties (Eiden et al., 1980). These results suggest that not only the amount of amino groups determine the adsorption properties of chitosan – various anions (sulphate/chloride) play a role (Mitani et al., 1995). The Co(II) and Ni(II) binding capacity of chitosan was also affected by the ionic strenght of anions. Sulphate ions strongly stimulated the metal binding ability of chitosan perls (Mitani et al., 1995; Becker et al., 2000). Lasko et al. (1993) analysed the binding capabilities of chitosan using different metals. In a pH 5 environment, heavy metal adsorption effectiveness to chitosan was put in the following order: Pb(II) > Fe(II) > Cd(II) > Cu(II). Chitosan strongly adsorbed copper. All of the used metals except lead were sulphate-linked. By using different modifications to create new chitosan derivates it was possible to efficiently to efficiently increase the adsorption qualities (Lasko et al., 1993; Guibal et al., 1998). Becker et al. crosslinked chitosan (DDA: 83 %) beads with a dialdehyde or a tetracarboxylic acid in order to obtain sorbents that are insoluble in aqueous acidic solution. The capacity for nickel(II), zinc(II) and cadmium(II) ions were measured in aqueous nitrate, chloride and sulphate solutions at pH 6. Four out of six chitosan derivates showed higher metal uptake rates in sulphate solution than in solutions of nitrate or chloride solutiuons. However, they possess poor metal ion selectivity, except in chloride solutions where cadmium(II) is preferentially bonded. In contrast, the other two chitosan derivates were highly selective for nickel(II) and cadmium(II). This seletive behaviour existed within a pH-range between pH 3 and pH 6 and was independent of the anion.

Heavy metals in oral cavity

In the dental medicine, amalgam has been used as a effective filling material for a long time. Amalgam consists of mercury (50 %), silver (40 %), tin (32 %), copper (30 %), zinc (2 %) and indium (5 %). The release of mercury ions (Hg2+) or gaseous mercury (Hg0) from the amalgam filling resulted in deterioration of human health (Harhammer, 2001; BfArM, 2003). Mercury contaminated food, e.g. fish and fish based products are another sources of mercury ingestions. The gastro-intestinal resorption ranges between 1 -10 %, wheras the pulmonary resorption amounts to about 80 % of ingestion (BfArM, 2003). Mercury (Hg 0+) resorption via the pulmonary tract leads to a direct mercury oxidation within the bloodstreem. Other ways of mercury uptake like dental pulp7, gingiva8 and oral mucosa are negligible (Harhammer, 2001).


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