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Biomedical Applications of Chitosan Chemically similar to both cellulose and hyaluronate, chitosan is gener- ally considered to be nontoxic 8 , biocompatible 5—6,9—10 , and biodegrad- able 6, It has several interesting biological properties that make it potentially useful in a wide range of biomedical applications. For example, chitosan agglutinates red blood cells, and is therefore under investigation as a hemostatic agent 8, Furthermore, because of its cationic nature and high charge density, orally administered chitosan binds fat in the intestine and may be effective in the treatment of hypercholesterolemia Chitosan also stimulates wound healing, and has been reported to perform better than conventional dressings for skin lacerations Finally, chitosan is being increasingly used for drug delivery, as in the controlled release of hormones over extended periods of time The potential for this material to serve as a cell scaffolding material for cartilage tissue engineering is currently under investigation, and in this area chitosan has several promising features.

Chitosan is a structural analog of the cartilage-specific glycosaminoglycans GAGs , which are known to be involved in regulating chondrocyte differentiation and biosynthesis and to mediate interactions between cells and between cells and the extracellular matrix. Thus, as noted by Suh and Matthew 16 , chitosan may be able to mimic certain biological activities of GAGs, including binding with growth factors and adhesion proteins.

The polycationic nature of chitosan facili- tates cell adhesion and allows for electrostatic interaction with anionic GAGs. As Madihally and Matthew 17 point out, ionic interactions with such negatively charged species could promote retention and organization of cartilaginous matrix constituents within a chitosan scaffold. Another interesting characteristic of chitosan is that oligosaccharide degradation products, liberated primarily by enzymatic hydrolysis of the acetylated resi- dues 18 , may stimulate the synthesis of cartilage-specific GAGs or be directly incorporated into these polysaccharides Finally, chitosan has antimi- crobial properties that could reduce the risk of bacterial infection when used as an implant material 20— Chitosan Variability As mentioned previously, the physical properties and biological response to chitosan depend strongly on its degree of deacetylation and mol wt.

Therefore, the choice of starting material deserves careful consideration. Because they are more crystalline, chitosan-based materials with a higher degree of deacetylation tend to absorb less moisture 6 , degrade more slowly 6,23 , and have higher tensile strength than materials that retain more acetamide groups 6.

It has also been reported that more highly deacetylated chitosan substrates support cell adhesion better than less deacetylated ones 1,5. Materials made from chitosan of higher mol wt exhibit greater tensile strength and moisture absorp- tion 4 , as well as higher fat binding 3 capacity compared to lower mol-wt preparations. Mol wt could also influence the release profile of drugs loaded into a chitosan cell scaffold 2.

The optimal chitosan formulation for a spe- cific tissue-engineering application can only be determined empirically. Com- mercial sources of chitosan include Vanson, Inc. Experimental Design One technique for the fabrication of bulk porous chitosan scaffolds has been presented by Madihally and Matthew Ice crystal nucleation and growth, as governed by the magnitude and directionality of thermal gradients, occurs upon freezing of a chitosan-acetic acid solution.

Subsequent lyophilization re- moves the ice phase and generates a porous material, and its overall porosity and mean pore size are modulated by the rate of freezing. The geometry of thermal gradients during freezing regulates pore orientation and connectivity. Thus, scaffold microstructure will depend on the shape of the mold used for freezing and on the freezer temperature.

The following are protocols for the synthesis of bulk porous chitosan scaf- folds suitable for tissue engineering applications and for the analysis of their surface morphology and porosity by scanning electron microscopy SEM and mercury intrusion porosimetry MIP , respectively.

MIP is based on the premise that a non-wetting liquid such as mercury will only intrude pores un- der pressure.

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MIP analysis can yield bulk density, apparent den- sity, total intrusion volume, total pore area, average pore diameter, and over- all porosity. Scaffold Fabrication 1.

Powdered chitosan. Magnetic stirrer and Teflon-coated stir bar. Cheesecloth optional. Polyethylene scintillation vial O. Waxed paper. Styrofoam insulator with approx 4 cm wall-thickness. Sharp forceps. Crushed dry ice and suitable container. Lyophilizer e. Lyophilization chamber to accommodate mL centrifuge tube.

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Sharp, straight-edged blades such as disposable microtome blades. Circular punch with 5—mm diameter. Phenol red-containing cell-culture medium. Scaffold Characterization Equipment 1. Scanning electron microscope. Mercury Intrusion Porosimeter e. Dissolve chitosan powder in 0. Using a magnetic stirrer and Teflon-coated stir bar, mix the solution for at least 48 h to ensure complete dissolution, at which point the pH of the solution should be approx 4.

Allow at least 24 h for freezing. Lyophilize samples for 24 h or until completely dry. Store dry samples in a dessicator until ready to rehydrate. Rehydrate scaffolds in a graded ethanol series see Note 1. Equilibrate the scaf- folds in two changes of each solution for at least 60 min see Note 3.

The ends of the sample should be discarded see Note 4. To produce a scaffold consisting only of material from the core of the original tube, use a sharp punch to cut a cylinder of 5—10 mm diameter from the thin slice. After this time, they should be treated as sterile. Before adding cells, confirm that there is no residual acidity by checking the color of the medium.

Scaffold Characterization see Note 8 3. Lyophilized samples require no further treatment. One 3-min coating is sufficient to eliminate charging inside the scope. Scan specimens in secondary electron detection mode. Mercury Intrusion Porosimetry MIP A detailed protocol for performing mercury intrusion analysis is beyond the scope of this chapter.


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However, a brief overview is provided. Degas the sample under vacuum and fill penetrometer sample holder with mercury. Proceed with low-pressure analysis. Allow at least 10 s equilibration time at each pressure step. A slight amount of shrinkage and distortion may occur during rehydration of lyophilized scaffolds. If lyophilized scaffolds are rehydrated in a neutral aqueous medium, they will become gelatinous and ultimately dissolve.

Hydrated scaffolds are soft, spongy, and highly extensible. The tensile elastic modulus of hydrated samples is in the range of — kPa 16 , and equilib- rium compressive modulus is approx 3—4 kPa. The use of ethylene oxide can also be considered as an alternate method of ster- ilization of lyophilized scaffolds.


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  8. The interior structure of scaffolds can be investigated using standard histological techniques. The use of hydrated scaffolds is recommended. Digitized images of sections prepared in this manner are suitable for quantitative analysis using soft- ware such as NIH Image. Chatelet, C. Biomaterials 22 3 , — Genta, I. Drug Dev. No, H. Food Sci. Nunthanid, J.

    Nanotechnology in Tissue Engineering

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