Seminar on Chitosan

Submitted by:Aditi Chandra MDS- 2012 Department of conservative dentistry and endodontics INTRODUCTION CHITIN is the second most ubiquitous natural polysaccharide after cellulose on earth. Often considered as cellulose derivative, even though it does not occur in organisms producing cellulose. Composed of (14)-linked D-glucose (N-acetylglucosamine) with a three dimensional -helical configuration stabilized by intramolecular hydrogen bonding. The principle derivative of chitin is CHITOSAN. Linear polymer of (14)-linked 2-amino-2-deoxy-D-glucopyranose. Is consequently a copolymer of N-acetylglucosamine and glucosamine. Therefore, chitin and chitosan are essentially the same polymer but with arbitrarily defined degrees of deacetylation (DD). If the DD is more than 40%, the term CHITOSAN is used.

HISTORY CHITIN was first discovered in mushrooms by French scientist Braconnot in 1811. It was also isolated from the cuticle of insect and named as CHITIN, which means envelop, i.e. exoskeleton of life in Greek, by French scientist Odier in 1823. De-acetylated chitin was discovered by Roughet in 1859, and named as CHITOSAN by German scientist Hoppe Seyler in 1894.

SOURCES Chitosan can be extracted from the shells of shrimp, crab, or lobster. It is also found in yeast and some fungi. Another inexpensive source of chitin is "squid pens," a byproduct of squid processing; these are small, plastic-like, inedible pieces of squid that are removed prior to eating.

MANUFACTURE Chitosan is commercially produced in different parts of the world (Japan, North America, Poland, Italy, Russia, Norway and India). A common method for the synthesis of chitosan is the deacetylation of chitin using sodium hydroxide in excess as a reagent and water as a solvent.The basic process for the manufacture of chitosan:1. Before the treatment, the shells are ground to make them more accessible.2. Removal of proteins and minerals such as calcium carbonate and calcium phosphate, by treatment with alkali and acid, respectively.3. After the completion of the manufacturing procedure, chitin is dried so that it can be stored as a stable intermediate for deacetylation to chitosan at a later stage.4. The process of deacetylation is achieved by treating chitin with a strong solution of sodium hydroxide at an elevated temperature.

PHYSIOCHEMICAL PROPERTIES

ParametersSpecification

AppearanceSmellViscosity (1% acetic acid soln.)Degree of DeacetylationBulk DensityLoss on Drying ( at 105oC)Ph Residue on IgnitionHeavy Metals(as lead)ArsenicTotal Plate CountPathogensParticle Size

White Flakes/PowderOdorless10-2000cps range65%- 90% ( as per customer requirement)0.15 to 0.7gm/ml ( as per customer requirement) Mg2+ [10 and 25 mM] (Tsai & Su, 1999).Age of a E. coli culture affects its interaction with chitosan, as bacterial cells, which are in the late exponential phase are most sensitive to chitosan (Tsai & Su, 1999).

Study on antimicrobial activity of chitosan with different molecularweights E. Coli and Staphylococcus aureus are used tostudy the antimicrobial activity ofchitosan of different molecular weights (MW). Theeffect of the concentration and MW of chitosan were investigated, respectively, and the antimicrobial mechanism was discussed. The antimicrobial activity of chitosan with MW below 305 kDa, was studied. As the concentration of chitosan increased,theantimicrobialeffectwasstrengthened. When the concentration reached 1.0%, the inhibition rate reached 100% for both E. coli and S. aureus. For S. aureus, a gram +ve bacteria, as the MW ofchitosanincreased, theantimicrobialeffect decreased. Themainreason mightbethechitosanof higherMWformsalm whichinhibitsnutrient adsorptions. For E.coli,agram-ve bacteria, asthe MWofchitosandecreased theantimicrobialeffect was enhanced. The main reason might be that the chitosan of lowerMWenters themicrobial cellmoreeasily, which disturbed the metabolism of the cell.

Susceptibility of Candida albicans and Enterococcus faecalis to Chitosan, Chlorhexidine gluconate and their combination in vitroAim: Analyse the sustain release of Chlorhexidine with Chitosan and to investigate the antimicrobial activity of 2% Chlorhexidine gel, 2% Chitosan gel and their combination against Candida albicans and Enterococcus faecalis.The study suggests that 2% Chlorhexidine gel in combination with 2% Chitosan gel has the highest antimicrobial effect against C. albicans and E. faecalis compared with 2% Chlorhexidine gel or 2% Chitosan gel alone.

Chitosan-EDTA new combination is a promising candidate for treatment of bacterial and fungal infections.Aim: Evaluate the antimicrobial activities of chitosan derivatives, EDTA, and the newly developed chitosan-EDTA combination against Gram-negative and Gram-positive bacteria as well as Candida albicans.Results: Indicated a synergistic antimicrobial activity of the new combination against Staphylococcus aureus and an additive effect against other microorganisms. Moreover, a short microbial exposure to chitosan EDTA combination (20-30 min) caused complete eradication.

PERIODONTITIS Periodontitis 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 (kinci 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 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. 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. In vivo, the tooth mobility and tooth pocket depths were significantly reduced.Clinical and Radiographic Evaluations of Chitosan Gel in Periodontal Intraosseous Defects: A Pilot StudyAim: Evaluate effects of chitosan on periodontal regeneration. Twenty chronic periodontitis patients were recruited. Following initial therapy, the patients were divided into four groups: group A, receiving chitosan gel (1% w/v); group B, receiving chitosan gel + demineralize bone matrix; group C: receiving chitosan gel + collagenous membrane; and group D, receiving flap only (control group). Clinical and radiographic measurements were recorded at baseline, day 90 (3rd month), and day 180 (6th month) after surgery. For clinical data, no significant differences were obtained among the treatment groups. Radiographic data revealed that except control group, all the other groups showed statistically significant bone fills indicating that chitosan gel alone or its combination with demineralize bone matrix/collagenous membrane is promising for periodontal regeneration.Application of Chitosan Gel in the Treatment of Chronic PeriodontitisAim: Evaluate the clinical effectiveness of chitosan, both as a carrier in gel form and as an active agent in the treatment of chronic periodontitis. The chitosan gel (1% w/w) incorporated with or without 15% metronidazole was prepared and applied adjunctive to scaling and root planing (SRP) in comparison to SRP alone (control group). Probing depth (PD), clinical attachment level, the amount of gingival recession, plaque index, gingival index, and gingival bleeding time index were recorded at baseline and at weeks 6, 12, and 24. In all groups, significant improvements were observed between baseline and week 24 (p < 0.05). No complications related to the chitosan were observed in patients throughout the study period. It is suggested that chitosan itself is effective as well as its combination with metronidazole in CP treatment due to its antimicrobial properties.

ANTI-PLAQUE AGENTA synergistic chlorhexidine/ chitosan combination for improved antiplaque strategiesBackground: The minor efficacy of chlorhexidine (CHX) on other cariogenic bacteria than mutans streptococcisuch as Streptococcus sanguinis may contribute to uneffective antiplaque strategies.Methods : In addition to CHX (0.1%) as positive control and saline as negative control, two chitosan derivatives (0.2%) and their CHX combinations were applied to planktonic and attached sanguinis streptococci for 2 min. The efficacy of the test agents on streptococci was screened by the following parameters: vitality status, colony-forming units (CFU)/ ml and cell density on enamel.Results: The first combination reduced the bacterial vitality to 0% and yielded a strong CFU reduction of 23 log units, much stronger than CHX alone. Furthermore, the first chitosan derivative showed a significant decrease of the surface coverage with these treated streptococci after attachment to enamel.Conclusions: Based on these results, a new CHX formulation would be beneficial unifying the bioadhesive properties of chitosan with the antibacterial activity of CHX synergistically resulting in a superior antiplaque effect than CHX alone.

Effect of water-soluble reduced chitosan on Streptococcus mutans, plaque regrowth and biofilm vitalityAim: Examine the effects of a newly developed water-soluble reduced chitosan on Streptococcus mutans, plaque regrowth, and biofilm vitality.Method: 1.0%, water-soluble reduced chitosan, with pH ranging from 6.0 to 6.5, molecular weights between 3,000 and 5,000 Da, and 70% degree of deacetylation, was used. To determine antibacterial and anti-plaque potency of chitosan, minimal inhibitory concentrations (MICs) for S. mutans and S. sanguinis short-term exposure to S. mutans, and clinical trial of plaque regrowth and biofilm vitality were conducted.Result: The water-soluble reduced chitosan exhibited potent antibacterial effect on S. mutans, and displayed a significant antibacterial and plaque reducing action during the 4-day plaque regrowth.

DENTAL CARIES

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. Y. Hayashi et al. published a study in the "Archives of Oral Biology" in 2007 that found individuals who chewed chitosan gum had less oral bacteria than those who did not chew the gum.

(Tarsi et al 1997) Chitosan has been shown to be an interesting candidate capable of preventing the adherence of Streptococcus mutans to hydroxyapatite in dental caries. This effect was attributed to the ability of chitosan to stimulate ordered regeneration of oral soft tissues, prevention of the deleterious effects of organic acids and bactericidal effects.

The desorptive effect of chitosan was weaker when Streptococcus mutans had adhered to the saliva coated hydroxyapatite in the presence of sucrose. Results: indicated the potential of the presence of minor amounts of chitosan in toothpastes, mouthrinses or chewing gums to impair the colonisation of the tooth surface

Chitosan microparticles for the controlled delivery of fluoride Objectives: To manufacture and characterise chitosan/fluoride microparticles prepared by spray drying and assess their utility as controlled release vehicles for fluoride. Conclusions: Bioadhesive chitosan/fluoride microparticles manufactured using a spray-drying protocol have been extensively characterised and further opportunity for optimisation identified. These microparticles may provide a means of increasing fluoride uptake from oral care products to provide increased protection against caries, however further work is required to demonstrate this principlein vivo.

HAEMOSTATIC AGENTChitosan is also found to have a haemostatic effect. Proposed to be due to an interaction between the cell membrane of erythrocytes and chitosan, and was independent of the classical coagulation cascade (Rao & Sharma 1997). 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, angiogenesis, reorganisation of the extracellular matrix, and granulation tissue have been demonstrated. Klokkevold et al (1999), found that topical application of chitosan to lingual incisions effectively decreased the intraoral bleeding time in a therapeutically anticoagulated (heparinised) rabbit model. Chitosan was able to facilitate lingual haemostasis, possibly through interaction with erythrocytes, linking them together to establish a cellular clot or a haemostatic plug. A study performed by R. Valentine et al., published in the "American Journal of Rhinology and Allergy," found that a chitosan gel applied to surgical wounds post-surgery significantly decreased bleeding time when compared to patients who did not have the gel applied. The Food and Drug Administration has approved the use of chitosan-treated bandages for controlling blood loss in emergency situations.Several chitosan-based wound dressings are available on the market for clinical use, including HemCon Bandage and ChitoFlex wound dressings (HemCon Medical Technologies, UK), as well as CELOX (Medtrade Products, England); both of which stated to be FDA approved.

ORTHOPEDIC AND CRANIOFACIAL IMPLANT DEVICESBumgardner 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.

CHITOSAN MONOMER PROMOTES TISSUE REGENERATION ON DENTAL PULP WOUNDSAim: Evaluate the applicability of chitosan monomer (d-glucosamine hydrochloride) as a pulp capping medicament. After 1 day, inflammatory cell infiltrations were observed to be weak when compared with the application of chitosan polymer. After 3 days, a remarkable proliferation of fibroblasts was seen near the applied chitosan monomer. The inflammatory cell infiltration had almost completely disappeared. After 5 days, the fibroblastic proliferation progressed, and some odontoblastic cells appeared at the periphery of the proliferated fibroblasts.These findings indicate that the present study is the first report that chitosan monomer acts as a biocompatibly stable medicament even at the initial stage of wound healing in comparison with the application of chitosan polymer.

EFFECTS OF CHITOSAN ON DENTAL BONE REPAIRAim: Assess effects of chitosan on socket repair after dental extraction.Method: Twenty four dental sockets of 15-24 year-old patients were visited by a maxillofacial surgeon for extracting premolar teeth for orthodontic purposes.

The sockets in one side were filled-in by chitosan. In the other side, the sockets were left unfilled. After 10 weeks, periapical radiographs were obtained and each socket was divided into coronal, middle and apical. Dental density of each socket in case and control groups was recorded. The density of regenerated bone was compared against the maximum bone density of each individual. Wilcoxon Singed-Rank test and paired t-test were used for data analysis.Results: Bone density in middle and apical sections in case group was significantly more than control group. In apical section in case group regenerated bone reached up to 98.2% of normal bone density. In each patient, the bone density in apical and middle sections was increase 29.3% and 10.8% of normal bone density.Conclusions: Chitosan significantly increased bone density in apical andmiddle sections. Chitosan can be used for bone repair in cases of bone loss.

CHITOSAN-BASED CEMENTSBioactivity of chitosan in dentistry. Preliminary data on chitosan-based cements.BACKGROUND: The chemical association ofchitosanwithinorganic salts, such ascalcium phosphate, finds a promising application in dentistry as room-temperature self-hardening cement.We present the physical, chemical and crystallographic characterization of newly-developed cements made of 1)calcium-phosphateand achitosangel obtained byacetic acidtreatment, and 2)calcium phosphateand achitosangel obtained byascorbic acidtreatment. Both cements are self-hardening at room temperature.METHODS: The cements were characterized by X-ray diffractography, scanning electron microscopy andfluorine-selective electrode analysis.RESULTS: Thechitosan-hydroxyapatitecements had hardness comparable to spongy boneCONCLUSIONS: The cements are promising for application in endodontics and restorative dentistry.

Properties of elastomeric calcium phosphate cementchitosan compositesIn the present study, chitosan was evaluated as the matrix for preparing CPC-chitosan composites.Methods: Cement specimens were prepared by mixing CPC powder (an equimolar mixture of tetracalcium phosphate and dicalcium phosphate anhydrous) with a chitosan solution at a powder/liquid ratio of 22.5Results: The CPCchitosan composites were more stable in water than conventional CPC. They did not disintegrate even when placed in water immediately after mixing. The CPCchitosan paste hardened within 10 min in all cases.Significance: This study demonstrates that CPCchitosan composites are stable in a wet environment and have acceptable mechanical strengths for clinical applications.

SMEAR LAYER REMOVALChitosan: a new solution for removal of smear layer after root canal instrumentationAim: To evaluate, by scanning electron microscopy (SEM), the efficacy of smear layer removal using chitosan compared with different chelating agents, and to quantify, by atomic absorption spectrophotometry with flame (AASF), the concentration of calcium ions in these solutions after irrigation.Method: The root canals of twenty-five canines were prepared using a crown-down technique and irrigated with 1% sodium hypochlorite.The teeth were randomly divided into groups (n=5), according to the type of final irrigation: 15% EDTA, 0.2% chitosan, 10% citric acid, 1% acetic acid and control (without final irrigation).Conclusions: 15% EDTA, 0.2% chitosan and 10% citric acid effectively removed smear layer from the middle and apical thirds of the root canal. 15% EDTA and 0.2% chitosan were associated with the greatest effect on root dentine demineralisation, followed by 10% citric acid and 1% acetic acid.

Time-Dependent Effects of Chitosan on Dentin StructuresObjective: Evaluate the effects of chitosan in dentins smear layer removal and erosion, in different time points and concentrations, by scanning electron microscopy (SEM) analysis.Method: Twenty canines were prepared by Crown-Down Technique and irrigated with 1% sodium hypochlorite. The teeth were randomly divided into ten groups, according to final irrigation type:G1 0.37% Chitosan (5 minutes), G2 - 0.1% Chitosan (5 minutes), G3 - 0.15% Chitosan (5 minutes), G4 - 0.2% Chitosan (5 minutes), G5 - 0.37% Chitosan (3 minutes), G6 - 0.1% Chitosan (3 minutes), G7 - 0.2% Chitosan (3 minutes), G8 - 0.37% Chitosan (1 minute) and G9 15% EDTA (3 minutes).The control group (G10) did not receive irrigation.Result: The results showed that the specimens irrigated with 0.1% chitosan solution showed the entire surface covered by smear layer, regardless of timing.The 0.15% chitosan group presented partially covering of smear layer on the surface with visible presence of few tubules and large amount of smear plug.The 0.37% chitosan group (5 and 3 minutes) caused excessive peritubular and intertubular dentinal erosion.Conclusion: Final irrigation with 0.2% Chitosan (3 minutes) provided the best results among all experimental groups and most similar to 15% EDTA in smear layer removal.

SPECIAL PRECAUTIONS & WARNINGSChitosan isPOSSIBLY SAFEfor most people when taken by mouth short-term (up to six months) or applied to theskin. When taken by mouth, it might cause mildstomachupset,constipation, or gas. Pregnancyand breast-feeding: Not enough is known about the use of chitosan during pregnancy and breast-feeding. Stay on the safe side and avoid use.Shellfish allergy: Chitosan is taken from the outer skeleton of shellfish. There is a concern that people with allergies to shellfish might also be allergic to chitosan.Warfarin (Coumadin) interacts with CHITOSAN: There is some concern that taking chitosan might increase the blood thinning effects of warfarin (Coumadin). Taking chitosan with warfarin (Coumadin) could increase the chance of bruising or bleeding. If you take warfarin, avoid taking chitosan.There is significant evidence that long-term, high-dose chitosan supplementation can result in malabsorption of some crucial vitamins andminerals includingcalcium,magnesium,selenium, andvitamins A,D,E, andK.Another possible risk of long-term ingestion of high doses of chitosan is that it could change the intestinal flora and allow the growth of unhealthful bacteria.Finally, there has been a case report of arsenic poisoning caused by long-term use of chitosan supplement

CONCLUSION The abundance of chitin and chitosan in nature and its safe toxicological properties has prompted researchers world-wide to investigate the potential applications of this unique biopolymer. As chitosan is principally obtained by partial deacetylation of chitin, the second most abundant natural polymer, a number of chitosan polymers varying in their physicochemical characteristics may be obtained by changing the degree of deacetylation. Chitosan possesses antibacterial effects enhances the wound healing by establishing optimal environmental conditions and is biocompatible and does not interfere with human immun system. These properties make it attractive for usage in dental medicine . In view of the above mentioned, varied and unique applications of chitosan and its derivatives, it may be pragmatic to say that these polymers have the potential to be used not only as pharmaceutical adjuvants for conventional or novel drug delivery systems, but also as biologically active compounds.

REFRENCESA. K. Singla and M. ChawlaChitosan: some pharmaceutical and biological aspects an updateJournal of Pharmacy and Pharmacology, JPP 2001, 53: 10471067.Mattioli Belmonte M et al. Bioactivity of chitosan in dentistry. Preliminary data on chitosan-based cements. Minerva Stomatologica1999, 48(12):567-576.Koide, S.S. (1998). Chitin-Chitosan: Properties, Benefits and Risks.Nutrition Research,8:6, 1091-1101.Lian-Ying Zheng, Jiang-Feng Zhu Study on antimicrobial activity of chitosan with differentmolecularweights Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China 29 July 2003CHITOSAN- WebMDDrugs.com: Complete Chitosan Information"Journal of Dentistry"; Chitosan Effect on Dental Enamel Demineralization: An In-Vitro Evaluation; T.M. Arnaud et al.; 2010"Archives of Oral Biology"; Chewing Chitosan-Containing Gum Effectively Inhibits the Growth of Carciogenic Bacteria; Y. Hayashi et al.; 2007"American Journal of Rhinology and Allergy"; The Efficacy of a Novel Chitosan Gel on Hemostasis and Wound Healing After Endoscopic Sinus Surgery; R. Valentine et al.; 2010L.E.S. FLAMINI et al. Time-Dependent Effects of Chitosan on Dentin Structures, Department of Restorative Dentistry, University of So Paulo - School of Dentistry of Ribeiro Preto, Ribeiro Preto, Brazil.Gemma M. Keegan et al. Chitosan microparticles for the controlled delivery of fluoride Journal of Dentistry Volume 40, Issue 3, March 2012, Pages 229240.K.V. Harish Prashanth,R.N. Tharanathan Chitin/chitosan: modifications and their unlimited application potentialan overview Trends in Food Science & Technology Volume 18, Issue 3, March 2007, Pages 117131.Pradip kumar dutta et al. chitin and chitosan: chemistry, properties and applications. Journal of scientific and industrial research vol. 63 JAN 2004 20-31.Bumgardner et al . Chitosan: Potential use as bioactive coating for orthopaedic and craniofacial/dental implants. J Biomater Sci Polymer Edn 14, 423-438.Duygu Boynueg et al Clinical and Radiographic Evaluations of Chitosan Gel in Periodontal Intraosseous Defects: A Pilot Study Journal of Biomedical Materials Research Part B: Applied Biomaterials November 2008.Hakan Akncbay et al. Application of Chitosan Gel in the Treatment of Chronic Periodontitis Department of Periodontology, Faculty of Dentistry, Hacettepe University, 8 March 2006Tsunenori Matsunaga et al. Chitosan monomer promotes tissue regeneration on dental pulp wounds Division of Cariology, Nagasaki University Graduate School of Biomedical Sciences,19 August 2005.Decker E-M, von Ohle C, Weiger R, Wiech I, Brecx M: A synergistic chlorhexidine/combination for improved antiplaque strategies. J Periodont Res 2005;40: 373377.N. V. Ballalet al. Susceptibility of Candida albicans and Enterococcus faecalis to Chitosan, Chlorhexidine gluconate and their combination in vitro Aust Endod J 2009; 35: 2933.K. Bae . E. J. Jun . S. M. Lee . D. I. Paik . J. B. Kim Effect of water-soluble reduced chitosan on Streptococcus mutans, plaque regrowth and biofilm vitality Clin Oral Invest (2006) 10: 102107.F. Ezoddini-Ardakan et al. Effects of chitosan on dental bone repairHealth 3 (2011) 200-205.