Introduction

Patients with burn wounds or other poorly healing wounds, like diabetes wounds, often suffer from various complications such as infections. Until now the common medical bandages have to be removed to monitor the wound healing progress. To examine if there is an infection, which implies the presence of pathogenic bacteria, samples of the wound have to be taken and studied in a specialized laboratory. This is a time-consuming and expensive process and we want to simplify and accelerate this procedure.

The solution for this problem is a hydrogel, with the advantages of the special characteristics of hydrogels such as biocompatibility, elasticity, and modifiable chemical properties. Most hydrogels can swell in aqueous solutions; for our purpose, as a wound bandage, it can be used to absorb some of the wound fluid [3][4]. Furthermore it can be attached hermetically and the moisture provided by the hydrogel therefore leads to ideal wound healing conditions [5]
An optimal polymer for such a hydrogel is the unique aminopolysaccharide chitosan. Beside beneficial properties like its biocompatibility, biodegradability, and film forming ability, chitosan has reactive amine side groups. This offers possibilities for modifications, like the linkage of a fluorophore to detect pathogenic bacteria (See Chemistry) [2].
The hemostatic chitosan is reported to have intrinsic antifungal, antibacterial, and antiviral properties [6]. Furthermore, it promotes scar free wound healing, has healing effects and acts antiallergic [7]. It is an ideal scaffold material to manufacture different types of hydrogels as salves, beads, sponges or solid bandages.

Production

To evaluate an ideal hydrogel, various compositions were tested, like pure chitosan or chitosan in combination with agarose, agar or alginate. The commercially available high-molecular weight chitosan was provided by Sigma-Aldrich (Munich, Germany). Due to their compounds, the hydrogels we wanted to create are not toxic, biodegradable, biocompatible, while at the same time being low-cost and simple manufacturing processes. They are easy to produce in different shapes and thicknesses. While being flexible they will not dissolve or disintegrate, which makes it comfortable for patients to wear. In addition to that, it is easy to manufacture, handle and use on patients. We were focused on working with basic laboratory equipment. For the preparation of chitosan hydrogels, an acidic environment is usually required to dissolve chitosan. Thus, the pH-level of our hydrogels are easy to regulate. We manufactured our chitosan containing hydrogel in aqueous acetic acid.
A long time of continuous mechanical stirring (6 h) is required to dissolve the chitosan in the acetic acid-deionized water solution. Under continuous pH measurement it needs mechanical stirring for 12 h. This solution needs to rest for at least 12 h for further processing.

Chitosan Hydrogel solidified in Alginate-Quercetin solution
Chitosan Hydrogel with high pH-Level
Chitosan-Agarose Hydrogel
Chitosan-Agar Hydrogel

Chitosan Hydrogel solidified in Alginate-Quercetin solution

The alginate/quercetin solution was poured into a mold and then liquid-frozen. The 2.2 % chitosan solution was then poured onto it and streaked out to the certain thickness, covered with the alginate/quercetin solution and placed into the 37 °C incubator for 24 h.

After the solidification process the arisen solid hydrogel was rinsed with ultrapure water.
Placed into an aqueous solution it will swell massively after time, but stays soft and pliable. The swelling degree of the hydrogel was calculated by

$\mathrm{DS\; =}\frac{\mathrm{Ww\; -\; Wd}}{\mathrm{<mn>Ww</mn>}}\mathrm{x\; 100}$

The hydrogels were saturated with the liquid in which it was immersed. Our manufactured hydrogel has a degree of swelling of almost 600%.

Chitosan-Agar Hydrogel

The mixture of chitosan with agar forms hydrogels with enhanced swelling compared to pure chitosan ones. Agar is a hydrophilic cell-wall polysaccharide extracted from the family of seaweeds. It composes of alternating (1-4)-D-galactose and (1-3)-3,6-anhydro-L-galactose repeating units and forms reversible gels even with a low concentration because of the formation of hydrogen bonds[2][10]. It is soluble in hot water and forms a gel during cooling. The polymer is biodegradable, low-cost, environmentally friendly and easy to extract. It is already used in pharmaceutical industry as a gelling, stabilizing and encapsulating agent [11]. It forms a stable, elastic gel which allows easy handling.

The shown hydrogel contains 1 % chitosan, 1 % acetic acid and 1 % agar. It is a very promising gel. It is a little bit adhesive to generate hermetically sealed, moist conditions for wound healing. Furthermore, it is more elastic than a gel with agarose.

Outlook

During the work with chitosan hydrogels various compositions were tested and promising hydrogels were produced. Hydrogels were manufactured with pure chitosan or chitosan in combination with agarose, agar or alginate. Depending on the concentration of chitosan and the respective gelling agent (agarose, agar, alginate), the gels were more or less solid. We manufactured stable and elastic gels which allow easy handling. According to the field of application a solid or smooth hydrogel is advisable.
The pH-level of the hydrogels is easy to regulate by rinsing it with a solution with the properly defined pH-level or using a higher or lower acetic acid concentration to dissolve the chitosan in an aqueous solution. This is beneficial for an adjustment to the respective wound and its pH-level.
Another advantage is the application for moist wound healing, as well as the factor that our hydrogels will seal the wounds airtightly. Here, the hydrogel prevents the formation of crusts and provides moisture. Both are important wound healing factors. The nutrient transport and release of signaling molecules is improved and scarring is massivly reduced with chitosan as wound toping layer. Our hydrogels can additionally deliver a monitoring bandage for affected patients without the need to disrupt the healing process and thus lighten the burden for the patients. The hydrogel should help to prevent or treat wound infections for example at any degree of burns. We thank the iGEM Team diagnost-X from Berlin for their provided expertise and informations regarding these medical problems.
The chitosan in itself or in combination other natural polymers is an ideal scaffold material to manufacture different types of hydrogels as salves, micro-, macroparticles, solid bandages and so on. Due to its wound healing effects and in combination with the medical benefits of a hydrogel it means an optimal wound healing dressing. Our hydrogels should be easy to load and therefore an ideal local slow-release drug-delivery vehicle for various therapeutic agents, pharmaceuticals, antimircobials or growth factors readily incorporated in situ to a treated tissue.

Group Picture

iGEM Technische Universität Darmstadt

We thank our sponsors, instructors and the iGEM organization!

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