Introduction

Patients with burn wounds or other poor-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 long time and expensive process and we want to simplify and accelerate this procedure.
The solution for the problem is a hydrogel, with the advantages of the special characteristics such as biocompatibility, elasticity, and modifiable chemical properties. Most hydrogels could 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 leads therefore to ideal wound healing conditions [5][Thanks to the iGEM Team diagnost-X from Berlin].
An optimal polymer for such a hydrogel is chitosan. Beside beneficial properties like biocompatibility, biodegradability, and film forming ability, chitosan has reactive amine side groups, which offer possibilities for modifications, like the linkage of a fluorophore to detect the pathogenic bacteria [2].
(see our Chemistry subproject)
The hemostatic chitosan is reported to have intrinsic antifungal, antibacterial, and antiviral properties [6]. Furthermore it promotes scar free wound healing and has care effects, and acts antiallergic [7]. It is an ideal scaffold material to manufacture different types of hydrogels, salves, pastes 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 hydrogels we wanted to create are due to their compounds not toxic, biodegradable, biocompatible, while at the same time having low-cost and easy to manufacturing processes. They are easy to produce in different sizes and thicknesses. While being flexible they keep their stability, which makes it comfortable for patients to wear, as well easy to manufacture, handle and apply on patients. The pH-level of our hydrogels are easy to regulate. We were focused to work with basic laboratory equipment. For the preparation of chitosan hydrogels, an acidic environment is usually required to dissolve chitosan. We manufactured our chitosan containing hydrogel in aqueous acetic acid.
A long time continuous mechanical stirring (6 hours) is required to dissolve the chitosan in the acetic acid-deionized water solution. Under continuous pH measurement it needs mechanically stirring for 12 hours. This solution needs to rest for at least 12 hours for further processing.

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, placed into the 37° incubator for 24 hours.

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

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

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

Chitosan Hydrogel with high pH-level

Manufacturing process of our chitosan hydrogel with a high pH-level was performed by rinsing the 2% chitosan solution with a defined NaOH solution with the desired pH-level.

Chitosan-Agarose Hydrogel

A hydrogel composed of chitosan and agarose fulfils most of the required criteria for an “ideal” wound dressing. Agarose is a biocompatible, linear polysaccharide which is extracted from marine algae. It consists of 1,4-linked 3,6-anhydro-α-L-galactose and 1,3-linked β-D-galactose derivatives.
When the agarose is dissolved in water, it forms a gel with a three-dimensional scaffold and a porous structure providing a good environment for cell adhesion, spreading and proliferation. It is capable of gelling within the desired site because of the polymer interaction. By varying the concentration of agarose in the hydrogel, the mechanical properties, which are similar to those of tissues, can easily be adjusted [8].
The preparation of such a hydrogel is quite simple, the components are dissolved in aqueous acetic acid and stirring until the solution is clear. The most promising variation was the hydrogel with 1 % agarose and 1 % chitosan. It forms a stable, elastic gel which allows easy handling. The simplicity of preparing the hydrogel and its multifunctionality allows many future applications of agarose-based hydrogels [9].

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].
The preparation is the same procedure as for agarose, just with agar instead of the agarose. The most promising variation was, as well as with agarose, the hydrogel with 1 % agar and 1 % chitosan. It forms a stable, elastic gel which allows easy handling.

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 allows 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 the proper pH-level solution or using a higher or lower acetic acid concentration to dissolve the chitosan in a aqueous solution. This is beneficial for an adjustment to the respective wound and the pH of the affected wound.
Another advantage is the application for moist wound healing, as well as the factor that our hydrogels could airtight the wounds. 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. The hydrogel should help to prevent or treat wound infections for example at any degree of burns. We thanks the iGEM Team diagnost-X from Berlin for their expertise and informations which they could provide us regarding these medical problems.
The chitosan with itself or in combination other natural polymers is an ideal scaffold material to manufacture different types of hydrogels as salves, pastes or solid bandages. Due to its wound healing effects and in combination with the medical benefits of a hydrogel it means an optimal wound healing dressing.

References

• Design: HTML5 UP