Determination of Fibrin Fiber Diameter Using Scanning Electron Microscopy and Image Processing Software

Abstract

Injectable, biodegradable scaffolds that mimic local tissue properties have great potential for aiding and accelerating the natural wound healing process. Ideally, scaffolds will have physical properties e.g., stiffness and microstructures, that are comparable to the tissue in which they will be applied. Fibrin is an insoluble protein that is formed in vivo during hemostasis via action of the enzyme thrombin on the soluble protein fibrinogen. It acts as a glue that holds together a loose platelet plug - a blood clot - that is degraded as wound healing progresses. The stiffness of this material can be easily tuned by adjusting its composition, as has been previously shown. These combined factors make fibrin a suitable scaffolding candidate for promoting cell delivery to wound sites, cell growth, and proliferation which are important parameters influencing the healing process. Scanning electron microscopy as well as other techniques are currently being leveraged in order to better understand fibrin's microstructure. Fibrin hydrogels are prepared in vitro by mixing fibrinogen, thrombin, and CaCl2 at various compositions. After additional processing, samples are then dried using either critical point drying or freeze-drying techniques to retain structure, and are subsequently sputter coated with gold/palladium, and imaged using a Hitachi SU7000. A MATLAB script was created that allows for the random selection and analysis of fibers. The resulting image is transferred into ImageJ where fiber sizes are measured. Average fiber diameter for fibrin gels prepared using fibrinogen at 6mg/ml and thrombin at 1U/ml is estimated to be 77nm.