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 Diagnostic-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 Chemistry
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 is 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. During this work various promising hydrogels could be produced. The kind of hydrogels we want 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, aswell 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-friezed. The 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 the hydrogels various compositions were tested, like pure chitosan or chitosan in combination with agarose, agar or alginate. We produced promising gels containing chitosan and agarose or agar. Depending on the concentration of chitosan and the respective gelling agent (agarose, agar, alginate) the gels were more or less solid. There were stable and elastic gels which allows easy handling or smooth gels like a crème. According to the field of application the solid or smooth hydrogel is advisable.
The pH-level of some of the hydrogels is easy to regulate by rinsing it with the proper pH-level solution. This is beneficial for an adjustment to the respective wound and the pH of this wound. The pH of some wounds for example should be reduced, which is easily possible with our hydrogel [Thanks to the iGEM Team Diagnost-x from Berlin for their help and this information].
Another advantage is the application for moist wound healing. Here the hydrogel prevents the formation of crusts and provides moisture. The nutrient transport and release of signaling molecules is improved and scarring is reduced.
The chitosan in combination with other natural polymers is an ideal scaffold material to manufacture different types of hydrogels, salves, pastes or solid bandages. Due to its wound healing effects and in combination with the benefits of a hydrogel, it provides the best supported wound healing for patients with poor-healing wounds.
It can deliver a monitoring bandage for these affected patients without the need to disrupt the healing process. The hydrogel should help preventing or treat wound infections of any degree of burns.

References

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