Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels

Abstract

Fibrin is a biopolymer that has been used in a variety of biomaterial, cell delivery and tissue engineering applications. The enzyme thrombin catalyzes the formation of fibrin microfibrils, which form a three-dimensional mesh in which cells can be directly embedded at the time of gel formation. In this study, fibrin hydrogels containing vascular smooth muscle cells were created using varying concentrations of thrombin. Over 7 days in culture, all gels decreased in volume as the fibrin matrix compacted, and the degree of gel compaction increased as thrombin concentration decreased. The material modulus and ultimate tensile stress of the gels also increased with decreasing thrombin concentration. Addition of thrombin to similar constructs made using collagen Type I did not show an effect on gel compaction or mechanical properties, suggesting that these effects were a result of thrombin's action on fibrin polymerization, and not cellular functions. Cell proliferation in fibrin hydrogels was not significantly affected by thrombin addition. Matrix examination using scanning electron microscopy showed increasing fibrin fiber diameters as thrombin concentration decreased. Confocal microscopic imaging of the actin cytoskeleton showed that cell morphology on twodimensional substrates of fibrin showed marked changes, with higher thrombin concentrations producing cells with longer cellular projections. However, these morphological changes were not as apparent in cells embedded in three-dimensional (3-D) matrices, in which cells exhibited a similar morphology independent of thrombin concentration. These results relate features of the matrix and cellular components of 3-D fibrin constructs to mechanical properties, and contribute to the understanding of structure-function relationships in cell-seeded, 3-D protein hydrogels.

Fibrin structural and diffusional analysis suggests that fibers are permeable to solute transport

“…4b-d). Overall, the results obtained in this study are consistent with those reported in literature [20,23,57,58]. For the same enzyme ratios (LL, HL, LH, HH), higher fibrinogen concentrations consistently gave higher G' values.…”

“…For the same enzyme ratios (LL, HL, LH, HH), higher fibrinogen concentrations consistently gave higher G' values. The effect of thrombin is not clear as contradictory findings have been reported in the literature; while a higher thrombin concentration decreased the material modulus in tensile tests of fibrin matrices encapsulating cells [58], a similar effect was only seen for higher thrombin to fibrinogen ratios (0.08 U/mg fibrinogen) in another study, with lower ratios having the opposite effect [20]. For the LH and HH compositions a different trend with thrombin concentration is seen for 5 and 20 mg/mL fibrinogen.…”

Regenerative potential of leucocyte‐ and platelet‐rich fibrin. Part B: sinus floor elevation, alveolar ridge preservation and implant therapy. A systematic review

“…L‐PRF contains a dense fibrin fibre network where platelets and leucocytes are enmeshed and it can serve as scaffold for other type of cells due to its favourable mechanical properties (Rowe et al. 2007, Khorshidi et al. 2016).…”

Hydrogel-based microfluidics for vascular tissue engineering

“…The physical properties of fibrin can be controlled in many ways. For example, the fibrinogen concentration, fibrinogen to thrombin ratio, Ca 2+ concentration, addition/ absence of Factor XIII, and pH conditions strongly regulate the network density of the fibrin gel [10]. However, fibrin gels undergo degradation and strong matrix remodeling in vitro, which results in construct shrinkage and weight loss [11].…”