3.4.1. Fibrin Clot Formation Time
Clotting time was determined using a stopwatch. The time was measured from the point when calcium chloride was added to the plasma at an interval of 10 seconds. The fibrin glue turned from a liquid to a semi-solid and then into a solid gel. To confirm the end-point, the Petri dish used to form the fibrin glue was slanted for a few seconds at an angle of roughly 45o at 10- second intervals. When the solid gel was observed, time was noted. Gelling of the contents occurred in less than three minutes to over sixty minutes depending on the calcium chloride concentration and the source of plasma.
3.4.2. Adhesion Test
Adhesion is a process by which an object or material adheres to another object or material. It is usually measured as a function of the mass that it can hold per unit surface area. Adhesion tests for fibrin glue can be performed differently using different methods. Typically, a tensile tester machine is used to determine the strength of fibrin glue. However, for this study, the adhesion test was performed using a custom machine that was developed for this purpose. It works on a similar analogy to that of a tensile tester.
Figure 3.4: Schematic diagram for custom adhesion measuring device(left) and using the device for measuring tensile strength on goat skin(right)
37 The adhesion performance of fibrin glue was tested on fresh goatskin collected from the neck or head region. The procedure for the test is as follows:
1. Fresh goatskin was cut into two slices, each of dimensions 5 cm by 1 cm.
2. The glue was prepared and applied to the bonding surface (1 cm by 1 cm) of two slices.
3. After bonding, the slices were allowed to stand for 30 min at room temperature (HAYASHI et al. 2014; Traver and Assimos 2006).
4. Then the bonded slices were pulled vertically by adding different weights to the weight holder starting from 5 mg to over 50 g. The weight at which the bond separated was divided by the bonded surface area of 1 cm2 to obtain the adhesion strength in gram per square centimeter (g/cm2).
3.4.3. Water Content Test
Water content is an important factor, which affects adhesion performance and is used to relate the variation of the adhesion performance to the plasma source. The water content test procedure is adapted from Murphy et al. (Murphy et al. 2010). The procedure for the test is as follows:
1. After the preparation of fibrin glue and the formation of a fibrin clot in Petri dishes, the gels were blotted gently to remove surface water and then weighed to obtain the initial weight (Wi).
2. Afterward, they were stored in a temperature-controlled oven at 37°C for 18 hours.
3. The Petri dishes were wrapped in a parafilm and some holes were made to control the rate of water vaporization. Afterward, the samples were lyophilized for up to 24 hours using a freeze-drying machine at 0.1 Pa pressure and -60oC and finally weighed to obtain the dry weight (Wd).
4. The water content was calculated using the formula below.
%Water content =
( – )× 100%
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3.4.4. Heat Curing Test
Inside the human body, the temperature is more or less regulated at around 37oC. To test the effect of heat on adhesion time, heat curing tests were performed. The procedure for the test is as follows:
1. Plasma solution from the same donor was taken into two Petri dishes. One of those Petri dishes was placed on a hot plate and the temperature of the plasma solution and the hot plate was monitored using a digital thermometer.
2. Once the petri dish on the hot plate reached 37oC, calcium chloride was added to the two Petri dishes at the same time and the stopwatch was started.
3. The gelling of the fibrin glue was monitored at an interval of 10 seconds as discussed previously in the clot formation time test. As soon as gelling occurred, the time was noted.
Figure 3.5: Heat Curing Test Setup
39
3.4.5. Cytotoxicity Test
Cytotoxicity tests are also called screening assays. They are used to evaluate the living cell’s reaction to the chemical or implant in a cell culture assay, including cell viability and the ability for cellular growth. Cytotoxicity tests were performed in the Centre for Advanced Research in Sciences, Dhaka University (CARS DU) using their facilities and services. Cytotoxicity tests were performed both qualitatively and quantitatively. The steps for the tests are as follows:
1. Vero cell line, kidney epithelial cells extracted from an African Green monkey, was maintained in DMEM (Dulbecco’s Modified Eagles’ Medium) containing 1%
penicillin-streptomycin (1:1) and 0.2% gentamycin and 10% fetal bovine serum (FBS).
2. Cells (2x104/100 µL) were seeded onto a 48-well plate and incubated at 37oC at 5%
CO2 for 24 hours.
3. On the next day, 25 µL of polymer solution (autoclaved) was added to each well.
4. Cell viability was examined after 48h of incubation using a trinocular microscope with a camera for qualitative analysis and a colorimetric cell proliferation assay kit (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, USA) following the manufacture’s protocol.
5. Duplicate wells were used for each sample.
3.4.6. SEM and EDS
To study the morphological changes in the fibrin cross-linked structure, a Scanning Electron Microscope (SEM, model JEOL JSM-7600F, Japan) was used. Moreover, Energy-Dispersive X-ray Spectroscopy (EDS) was also performed to determine the concentration of various chemicals in various locations of the cross-linked polymer structure.
1. Freeze-dried samples were used for SEM. The samples with various concentrations of calcium chloride and polymer solutions were freeze-dried for this purpose.
40 2. The Petri dishes containing prepared glue were wrapped in a parafilm and some holes were made to control the rate of water vaporization. Afterward, the samples were lyophilized for 24 hours using a freeze-drying machine at 0.1 Pa pressure and -60oC.
3. The samples were gold-sputtered in a vacuum chamber. Gold sputtering improves image quality for biological samples.
4. The gold-sputtered samples were placed inside the SEM device and the images were taken at different magnification levels. Afterward, EDS data was generated by fixing location spots.
5. The SEM images were analyzed to determine any observable difference in the structure of the fibrin glues due to the different concentrations of calcium chloride and the effect of various polymers and their concentration.
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RESULTS AND DISCUSSION
To determine the performance of the developed fibrin glue, several tests were conducted and their results were analyzed and compared with commercial fibrin adhesives. This chapter discusses the results obtained in this research study. In the first half of this chapter, a qualitative analysis of the fibrin glue formation is discussed with relevant images. In the later part of this chapter, the results of the characterization tests like clotting time, adhesion strength, water content, cytotoxicity, SEM, and EDS are discussed.