Introduction

Alginate Silk-Fibroin is a prospective  biomaterial that has numerous properties. It  structure consists of a protein fiber  composed of linked amino acids in a  secondary sheet structure. This secondary  scaffold consists of pleated beta sheets. This  creates a unique material that is biocompatible and able to be degraded via  naturally occurring enzymes within the body  such as chymotrypsin, actinase, and carboxylase. These properties make it a  prospective material for use in medical and  pharmaceutical applications.

Alginate silk fibroin has numerous uses  within the medical sector, one of which is  coagulation. Silk scaffolds have shown  hemostatic properties by increasing platelet  formation rates by absorbing platelets and  helping them release clotting factors.  

Both sodium alginate and silk have been  shown to increase wound healing and  decrease overall wound inflammation. Hence  making a synergistic relationship that is ideal  for wound dressings.

Activation of Bodily Clotting Systems

The human clotting system  is composed of 2 parts, the  intrinsic clotting system and  the extrinsic clotting  system.  The intrinsic clotting  system is activated via  clotting factors XII and  high-molecular-weight  kininogen. While the  extrinsic system is activated  via external damage  mediated via tissue factor.

Alginate silk fibroin has 2 methods of increasing the rate of clotting:  

1. Chemical stimulation: Activation of the intrinsic clotting system via platelet stimulation  

1. Physical enhancement: Direct pressure via swelling of sodium alginate, reducing overall blood flow into the wound.

Silk Fibroin Synthesis via CaCl2

The synthesis of Silk-Fibroin utilizes a novel method. Instead of using LiBr as a solvent, CaCl2 is used. This results in a faster synthesis of silk-fibroin, cutting down on both time and money.

Silk Sample Preparation

The following method was observed:

1.  Washed with 5ml of methanol for sterilization.

2. Rinse with 10ml sterile, diluted PBS.

3. Rinsed with sterile water then excess water removed using pippette.

4. Dried using vacuum oven at 30C over 1 hour.

Sample Preparation

Blood sample were processed utilizing the following method:

1.  Sample that were obtained from local market then freeze-dried prior the use. 

2. Analyzed using a Giemsa stain to ensure RBC and platelet integrity.

3. 0.1% DMSO is titrated into the sample (final ratio of DMSO: sampe is 10:1).

4. Sample is shaken and incubated at 10C ice bath.

5. Sample is agitated, then flash-frozen.

Baseline and Maximum Release Preparation

I. To prepare maximum release (no clot formation):

1.  add 28 uL of sample and 2ml of water.

2. Agitate manually for 5 minutes.

3. Analyze absorbance at 200-1000 nM

II. To prepare baseline (for both):

1.  Pulverize silk fibroin matrix

2. Mix with water.

3. Agitate the mixture and leave for 30 minutes.

4. Transfer to an eppendorf and centrifuge.

5. Transfer the mixture to cuvette and take baseline measurement.

UV-VIS Analysis and Results

From the graphs, one can deduce several observations:

1.  The release of hemoglobrin is done in a non-linear fashion.

2. Higher clotting capabilities can be seen in silk-fibroin compared to regular cotton spun-bound gauze.

3. Absorbance was highest at 570-580 nM range due to the use of oxygenated blood as compared to deoxygenated blood (556 nM).

4. Peak flattening in graph 3 suggest some sort of increased hemostatic ability.

*Look at the image and here's the Graph legend:

• Graph 1: shows the absorption of SF over the 530-580 nM.

• Graph 2: indicates the absorbance peak at a wavelength of 575 nM over 45 minutes.

• Graph 3: is whole absorbance over the spectrum of SF-Alginate Matrix.

Conclusion

To conclude, it is likely that SF-alginate matrix is able tointeract with blood that can increase clotting  rates and reduce mortality rates in massive hemorrhagic injuries. The data collected thus far is still  deemed as preliminary. Silk-fibroin is also seen to be superior at clotting compared to both regular  cotton gauze and Quikclot. Further research is recommended to further quantify the clotting abilities of  SF-Alginate matrix, such as utilizing a fibrinogen assay to get more accurate results.