Eugene Khor

Implantable applications of chitin and chitosan.

Chitin, extracted primarily from shellfish sources, is a unique biopolymer based on the N-acetyl-glucosamine monomer. More than 40 years have lapsed since this biopolymer had aroused the interest of the scientific community around the world for its potential biomedical applications. Chitin, together with its variants, especially its deacetylated counterpart chitosan, has been shown to be useful as a wound dressing material, drug delivery vehicle and increasingly a candidate for tissue engineering. The promise for this biomaterial is vast and will continue to increase as the chemistry to extend its capabilities and new biomedical applications are investigated. It is interesting to note that a majority of this work has come from Asia. Japan has been the undisputed leader, but other Asian nations, namely Korea, Singapore, Taiwan and Thailand have also made notable contributions. More recently, China has joined the club to become an increasingly major research source for chitin and chitosan in Asia. This review surveys select works of key groups in Asia developing chitin and chitosan materials for implantable biomedical applications.

Methods for the treatment of collagenous tissues for bioprostheses

Collagenous tissue as a biomaterial possesses many favourable characteristics and advantages over synthetic materials. The resemblance to human tissue suggests that it has a performance advantage over alternative materials. This advantage has been exploited to produce clinical devices that have been implanted in patients for more than a quarter of a century. The method of treating collagenous tissue for bioprostheses has developed from crude exposure of tissue to chemicals to a sophisticated level of considering the biochemical, chemical, engineering and clinical aspects of the process. This review focuses on the various chemical and physical treatments that have made the bioprostheses possible, highlighting the chemical agents and the cross-linking mechanism involved.

The degree of deacetylation of chitosan: advocating the first derivative UV-spectrophotometry method of determination

The degree of deacetylation (DD) is increasingly becoming an important property for chitosan, as it determines how the biopolymer can be applied. Therefore, a simple, rapid and reliable method of determining the DD for chitosan is essential. In this report, the DD of chitosan was determined by nuclear magnetic resonance (NMR), linear potentiometric titration (LPT), ninhydrin test and first derivative UV-spectrophotometry (1DUVS). The DD was calculated on a per mol basis instead of on a per mass basis. This is important as the molecular weights of N-acetyl-d-glucosamine and d-glucosamine are different. By converting the mass of N-acetyl-d-glucosamine and d-glucosamine into mols and calculating for the percentage of d-glucosamine present in the chitosan sample, a more accurate estimation of the DD can be obtained. Of the four methods, there is good correlation between 1DUVS and NMR. The concentration of chitosan solution for 1DUVS analysis was standardised as 0.1000 mg chitosan per ml of 0.0100 M acetic acid solution. The presence of d-glucosamine was corrected for by a reference curve for N-acetyl-d-glucosamine. 1DUVS is easy to perform, sensitive and the interference of other contaminants to the results is minimal compared with the other three methods. Therefore, we advocate 1DUVS to be used as the standard methods for routine determination of DD of chitosan.

Wound Dressing with Sustained Anti-Microbial Capability

To overcome current limitations in wound dressings for treating mustard-burn induced septic wound injuries, a nonadherent wound dressing with sustained anti-microbial capability has been developed. The wound dressing consists of two layers: the upper layer is a carboxymethyl-chitin hydrogel material, while the lower layer is an anti-microbial impregnated biomaterial. The hydrogel layer acts as a mechanical and microbial barrier, and is capable of absorbing wound exudate. In physiological fluid, the carboxymethylated-chitin hydrogel swells considerably, imbibing up to 4 times its own weight of water and is also highly porous to water vapor. The moisture permeability of the dressing prevents the accumulation of fluid in heavily exudating wounds seen in second-degree burns. The lower layer, fabricated from chitosan acetate foam, is impregnated with chlorhexidine gluconate. From the in vitro release studies, the loading concentration was optimized to deliver sufficient anti-microbial drug into the wound area to sustain the anti-microbial activity for 24 h. The anti-microbial activity of the dressing against Pseudomonas aeruginosa and Staphylococcus aureus was tested using the Bauer-Kirby Disk Diffusion Test.

The chitosan yield of zygomycetes at their optimum harvesting time

Fungi are a promising alternative source of chitosan. Fungi can be manipulated to give chitosan of more consistent and desired physico-chemical properties compared to chitosan obtained from crustacean sources. Chitosan was extracted from the mycelia of Rhizopus oryzae USDB 0602 at various phases of growth. The growth phase which produced the most extractable chitosan was determined to be the late exponential phase. In contrast to previous work on the screening of chitosan from fungal sources, mycelia of the fungi used in this study were harvested at their late exponential growth phase instead of at a fixed incubation time. The amount of extractable chitosan varied widely among the fungal strains. Gongronella butleri USDB 0201 was found to produce the highest amount of extractable chitosan per ml of substrate, followed by Cunninghamella echinulata and Gongronella butleri USDB 0428. However, in terms of yield of chitosan per unit mycelia mass, C. echinulata was the best strain among all fungi in the experiment. Therefore, besides G. butleri USDB 0201, C. echinulata can also be considered to be used in the commercial production of chitosan.

Hydroxyapatite–chitin materials as potential tissue engineered bone substitutes

Hydroxyapatite (HA) in 25%, 50% and 75% w/w fractions was incorporated into chitin solutions and processed into air- and freeze-dried materials. These HA-chitin materials were exposed to cell cultures and implanted into the intramusculature of a rat model. The HA-chitin materials were found to be non-cytotoxic and degraded in vivo. The presence of the HA filler enhanced calcification as well as accelerated degradation of the chitin matrix. The freeze-dried HA-chitin matrixes were selected for further cell seeding experiments because of their porous nature. Mesenchymal stem cells harvested from NZW rabbits were induced into osteoblasts in vitro using dexamethasone. These osteoblasts were cultured for 1 week, statically loaded onto the porous HA-chitin matrixes and implanted into bone defects of the rabbit femur for 2 months. Histology of explants showed bone regeneration with biodegradation of the HA-chitin matrix. Similarly, green fluorescence protein (GFP) transfected MSC-induced osteoblasts were also loaded onto porous HA-chitin matrixes and implanted into the rabbit femur. The results from GFP-transfected MSCs showed that loaded MSCs-induced osteoblasts did not only proliferate but also recruited surrounding tissue to grow in. This study demonstrates the potential of HA-chitin matrixes as a good substrate candidate for tissue engineered bone substitute.

Concurrent production of chitin from shrimp shells and fungi

Crustacean shells constitute the traditional and current commercial source of chitin. Conversely, the control of fungal fermentation processes to produce quality chitin makes fungal mycelia an attractive alternative source. Therefore, the exploitation of both of these sources to produce chitin in a concurrent process should be advantageous and is reported here. Three proteolytic Aspergillus niger (strains 0576, 0307 and 0474) were selected from a screening for protease activity from among 34 zygomycete and deuteromycete strains. When fungi and shrimp shell powder were combined in a single reactor, the release of protease by the fungi facilitated the deproteinization of shrimp-shell powder and the release of hydrolyzed proteins. The hydrolyzed proteins in turn were utilized as a nitrogen source for fungal growth, leading to a lowering of the pH of the fermentation medium, thereby further enhancing the demineralization of the shrimp-shell powder. The shrimp-shell powders and fungal mycelia were separated after fermentation and extracted for chitin with 5% LiCl/DMAc solvent. Chitin isolates from the shells were found to have a protein content of less than 5%, while chitin isolates from the three fungal mycelia strains had protein content in the range of 10-15%. The relative molecular weights as estimated by GPC for all chitin samples were in the 10(5) dalton range. All samples displayed characteristic profiles for chitin in their FTIR and solid-state NMR spectra. All chitin samples evaluated with MTT and Neutral Red assays with three commercial cell lines did not display cytotoxic effects.

γ Irradiation of chitosan

Chitosan has potential biomedical applications that may require the final products to be sterilized before use. The γ irradiation of purified and highly deacetylated chitosan fibers and films at sterilizing doses (up to 25 kGy) caused main chain scissions. The viscosity average molecular weight of the polymer decreased with increasing irradiation dose, the radiation yields of scission being 1.16 in air and 1.53 in anoxia. Preirradiation application of a negative pressure of 100 kPa disrupted the network structure, which may have contributed to the greater radiation yield obtained by chitosan fibers in anoxia. Radiation induced scission of the chitosan chains resulted in a lower glass transition temperature (Tg), indicative of higher segmental mobility. The Tg was below ambient at an irradiation dose of 25 kGy in air. Irradiation in air improved the tensile strength of the chitosan film, probably due to changes in chain interaction and rearrangement. Irradiation in anoxia did not affect film properties significantly, partly because the preirradiation application of negative pressure had a negligible effect on the structure of the chitosan film. Polymer network structure and the irradiation conditions are therefore important determinants of the extent of radiation induced reactions in chitosan.

Preparation of a chitin-apatite composite by in situ precipitation onto porous chitin scaffolds.

Composites of chitin with calcium phosphate were obtained by in situ precipitation of the mineral from a supersaturated solution onto chitin scaffolds. The chitin scaffolds were obtained by freeze drying to give a highly porous structure possessing a polar surface favorable for apatite nucleation and growth. THe extent and arrangement of calcium phosphate deposits on the chitin and substituted chitin scaffolds were explored. Up to 55% by mass of calcium phosphate could be incorporated into chitin scaffolds. Deposits on the chitin surface were a continuous apatite carpet nature while deposits on carboxymethylated chitin surfaces displayed a spherical morphology. Carboxymethylation of chitin exerts an overall inhibitory effect towards calcium phosphate deposition, but it provides for site-specific nucleation of the mineral phase. In situ precipitation can be an important route in the future production of various polymer-calcium phosphate composites.

Preparation and characterization of chitin beads as a wound dressing precursor

Chitin was dissolved in N, N-dimethylacetamide/5% lithium chloride (DMAc/5%LiCl) to form a 0.5% chitin solution. Chitin beads were formed by dropping the 0.5% chitin solution into a nonsolvent coagulant, ethanol. The beads were left in ethanol for 24 h to permit hardening, consolidation, and removal of residual DMAc/5%LiCl solvent in order to give spherical chitin beads uniform size distribution. The ethanol-gelled chitin beads had an average diameter of 535 microm. The chitin beads were subsequently activated in 50% (w/v) NaOH solution and reacted with 1.9 M monochloroacetic acid/2-propanol solution to introduce a carboxymethylated surface layer to the chitin beads. The bilayer character of the surface-carboxymethylated chitin (SCM-chitin) beads was verified by Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), and confocal microscopy. The bilayered SCM-chitin beads were found to absorb up to 95 times their dry weight of water. These SCM-chitin beads have potential as a component of wound dressings.