Team:METU-Gene/Collagen Sponge



 The release rate of bioactive hEGF from crosslinking collagen sponges

The microstructure and the drug release rates of collagen sponges were modified through treatment with different types (glutaraldehyde (GTA), genipin and 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC)), different concentrations of crosslinking agents and various preparation conditions.

A good correlation was obtained for in vitro release rates of rhEGF using the power model. The crosslinked rhEGF collagen sponges showed a successful delivery of rhEGF in bioactive form to stimulate cell proliferation.

In addition, EGF can inhibit gastric acid secretions in the stomach, enhance the proliferation and keratinization of epithelial tissues and accelerate wound healing. Due to its wound healing properties, EGF is an attractive candidate for a therapeutic drug. Studies have demonstrated that topical applications of EGF promote wound healing in healthy and impaired healing animals.

Since Carpenter and co-workers first reported that for a mitogenic effect of EGF, a continuous exposure of the target cells to EGF was required for a minimum of 6–12 h, maintaining an effective topical concentration at the wound site for a certain period of time has become vital in the application of EGF. Indeed, we increased this continuous exposures by using Quaroum Sensing Mechanism of E.coli.

Collagen is a major constituent of the connective tissue and is potentially a highly useful biomaterial. It has characteristics that are suitable in medical application, such as biodegradability and weak antigenicity, and it has been used in resorbable surgical sutures, hemostatic agents, and wound dressings for many years.

An in vitro controlled release study was conducted to investigate the mechanism of recombinant human epidermal growth factor (rhEGF) release from the different degree of crosslinked collagen sponges.

Characterization of the rhEGF-collagen sponges
[1]Determination of the degree of crosslinking

The crosslinking degree could then be obtained from the differences between the absorbance values before and after the crosslinking. The equation is as follows:

http://2009.igem.org/wiki/images/9/9b/Formul1.jpg

where s is the sample and ncl is non-crosslinked.

[2]Water-binding capacity

The water uptake of the collagen sponges was calculated using the following equation:

http://2009.igem.org/wiki/images/a/a3/Formul2.jpg

where Wd is the weight of the dry sponge and Ws is the weight of the swollen sponge.

[3]Release kinetics

To determine the possible release mechanism, drug release from collagen sponges was fitted to the following power model:

http://2009.igem.org/wiki/images/6/64/Formul3.jpg

where Mt/M is the fractional drug release percentage at time t, and k is a constant related to the properties of the drug delivery system and n is the diffusional exponent which characterizes the drug transport mechanism.

Recombinant hEGF release from collagen sponges
Figure 1 shows the release profiles of rhEGF from collagen sponge at 37 �C in PBS with/without collagenase solution.

http://2009.igem.org/wiki/images/8/8e/Figure1.jpg

Chih-Hui Yang in his study supposed that under the in vitro non-degradation conditions, rhEGF was initially released by diffusion. Generally speaking, since collagen is enzymatically degraded, low final release values are expected in the absence of any enzymes. Therefore, collagenase was employed for the model of the in vitro rhEGF release study. In project, this case is also valuable.

Therefore, the influence of the types and the concentrations of the crosslinking agents and the preparation conditions on the structures and characteristics of collagen sponges, and the rhEGF release from collagen sponges were compared in his study.

Three different types of crosslinking agents, GTA, genipin and ECD were used to prepare crosslinked collagen sponges. The rhEGF release patterns from collagen sponges are shown in Figure 2.

http://2009.igem.org/wiki/images/b/b0/Figure2.jpg

The drug release rate from crosslinked collagen sponges treated with EDC was the fastest, followed by collagen sponges treated with genipin and GTA, respectively. The EDC crosslinked collagen showed no release control effect, which was probably due to the fact that EDC increased the water-solubility and lowered the viscosity of collagen (data not shown). GTA crosslinked collagen showed the most potent release control effect than the other two (EDC and genipin). However, since we want controlled and orderly release system which will be improved our transgenic bacteria, we used genipin for formation our cellulose Wound Dressing layer in three different types of crosslinking agents, GTA, genipin and ECD.

Preparation of Collagen Sponge
•	Solve 48,8 µg collagen in 6,5 ml 0,05 M acetic acid to prepare 0,75 % collagen solution (the final concentration of collagen solution is 7,5 mg/ml)

http://2009.igem.org/wiki/images/6/62/11.jpg

•	Overnight the collagen solution at 350 C with magnetic heater to dissolve the collagen in acetic acid completely

http://2009.igem.org/wiki/images/1/13/22.jpg

•	Dissolve 10 mg genipin (cross-linker for collagen) in 1 ml 70 % ethanol (the concentration of genipin solution is 1 %) •	Add 650 µl genipin solution to the collagen solution (final concentration of genipin solution in the collagen solution is 0,1 %) •	Place 1 ml prepared final solution to well

http://2009.igem.org/wiki/images/d/da/33.jpg

•	Wait the prepared solution in the wells for 48 hours at room temperature

http://2009.igem.org/wiki/images/9/98/44.jpg

•	Keep the wells at -800 C at 24 hour •	Lyophilize the frozen solution

http://2009.igem.org/wiki/images/8/86/55.jpg

Conclusion
Crosslinked rhEGF-collagen sponges can be useful for controlling the release of rhEGF. Results have shown that upon increasing the amount of genipin or GTA or EDC, the microstructure of collagen sponges becomes more rigid, and the hydrophilicity is reduced, resulting in a decreased drug release rates and an increased water uptake. A good correlation was obtained for in vitro release rates of rhEGF from crosslinking collagen sponges using the power model.