Fabrication of Fibrin Fibres to Guide Cellular Alignment

Abstract

Many tissues have a distinctly aligned microstructure which is critical for their optimal function such as muscle tissue and vasculature. Therefore, in order to regenerate or model those tissues, tissue engineering constructs need to be able to mimic their native cellular alignment and orientation. In addition to mimicking the alignment, the three-dimensionality of tissues needs to be considered in order to obtain more physiologically accurate solutions. Biopolymer fibres and hydrogels have emerged as promising materials due to their versatility and tailorable properties. Fibres can be used to prepare organised constructs that mimic fibrous tissues and to guide cellular alignment. Hydrogels, on the other hand, are excellent materials for creating a three-dimensional growth environment for cells due to their permeability and capability to mimic the native extracellular environment. Fibrin is a fibrous polymer formed from the plasma glycoprotein fibrinogen by the action of the serine protease thrombin. Fibrin is a highly appealing material for tissue engineering since it can promote cell attachment, migration and proliferation. In addition, it has unique mechanical properties, such as viscoelasticity and strain hardening. Previously, fibrin fibres have been successfully produced by extrusion and their ability to guide cellular alignment has been studied. However, in only two studies, fibrin fibres have been placed inside a hydrogel. In those studies, fibre encapsulation was noticed to affect the behaviour of cells enclosed inside fibres. Also, cell alignment was limited to cells close to fibres when encapsulated in fibrin gel matrix. The objective of this thesis was to fabricate fibrin fibres with a simple extrusion method and to investigate the capability of the fibres to provoke cellular alignment. In addition, the physical and mechanical properties of the fibres were characterised. All polymers used in this thesis were prepared using published methods. A gelation test and an initial extrusion test were conducted to find the optimal fibrin composition for fibre production and cell culture. Changes in fibre diameter after various processing steps were monitored using a light microscope, by measuring the diameter of the fibres. Fibre swelling and stability was studied by incubating the fibres in medium as such and between two layers of gellan gum-gelatin hydrogel. Alignment of the fibrin fibril network, due to stretching during the fabrication process, as well as the morphology and surface structure of the fibres was examined with a scanning electron microscope (SEM). A tensile test was conducted to characterise the mechanical properties of the fibres. Cell alignment was studied by culturing WI-38 fibroblasts on fibrin fibres placed on top of gel-lan gum-gelatin hydrogel as such or when encapsulated in the gel. The results show that fibrin fibres were successfully fabricated with different compositions. Stretching during the fabrication process significantly decreased fibre diameter, however, less than anticipated due to high elasticity of fibrin. The produced fibres had good stability since they stayed intact during long-term incubation. Also, the fibres did not swell back to their initial wet, stretched diameter upon rehydration. SEM images of the fibres showed clearly aligned fibrils albeit there were differences in the degree of alignment between different areas. In terms of mechanical properties, the fibrin fibres were highly extensible and elastic. Also, despite a manual fabrication method there was no big difference between the measured fibres. Cell culture studies with fibroblasts showed that the fibres were capable of provoking cellular alignment. However, this phenomenon was observed only when the cells were in direct contact or close vicinity of them. Therefore, alternative cell seeding methods and culture setups need to be considered to further improve the outcome. In conclusion, fibrin fibres with a longitudinally oriented fibril network could be easily fabricated with the simple extrusion method. The obtained fibres were highly extensible and elastic. Cellular alignment was observed when the cells were in close proximity of the fibres.