Influencing biophysical properties of fibrin with

Authors: Esther Potier Jérôme Noailly Christoph M Sprecher Keita Ito

Publish Date: 2010/01/26

Volume: 45, Issue: 9, Pages: 2494-2503

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Abstract

Fibrin has been proposed as cell scaffold for numerous tissue engineering applications While most of the studies have focused on fibrinogen and thrombin other components of fibrin can also affect its properties The present study aimed to evaluate the effects of buffer solution composition on fibrin biophysical properties Fibrin scaffolds were synthesized with different calcium chloride and factor XIII FXIII final concentrations Light transmission was determined as a relative semiquantitative estimator of fiber structure differences and two compositions resulting in translucent and opaque gels were tested for mechanical and biological properties Gels were seeded with mouse mesenchymal cells C3H10T1/2 or bovine bone marrowderived mesenchymal stromal cells and cultured up to 10 or 24 days before cell number morphology and distribution were evaluated Calcium increased gel opacity ie fiber thickness while chloride and FXIII decreased it Opaque gels displayed a fluidlike viscous behavior while translucent gels showed improved elastic properties Both compositions supported survival of both cell types with opaque gels leading to better proliferation but significant scaffold shrinkage after 17 days of culture These results demonstrated that calcium chloride and FXIII modulate the biophysical properties of fibrin and can be used to adjust mechanical and biological properties for tissue engineering applicationsFibrin glues are biological hydrogels reproducing the final stages of the clotting cascade the conversion of the soluble plasma fibrinogen into insoluble fibrin by the action of thrombin 1 Since many years fibrin glues have been widely used as surgical sealants More recently they also have been proposed as scaffolding for cell delivery in several tissue engineering applications 2 Numerous in vitro studies indeed have demonstrated the ability of fibrin gels to support survival proliferation and/or differentiation of many cell types 3 4 5 6 including mesenchymal stromal cells MSCs 7 8 9This aptitude to sustain cell growth is regulated in part by the fibrin gel composition Fibrin glues usually consist of two constituents highly concentrated fibrinogen and thrombin 10 The fibrinogen buffer solution also includes factor XIII FXIII antifibrinolytic agent aprotinin or tranexamic acid fibronectin and traces of other plasma proteins such as plasminogen Thrombin buffer solution on the other hand contains calcium and sodium chlorides High fibrinogen and to a lesser extent low thrombin concentrations have been shown to reduce the proliferation of human MSCs 7 8 9 and dermal fibroblasts 3 The fibrinogen concentration thresholds determined by these studies however varied from 18 to 50 mg/mL fibrinogen This high variability in cell behavior in response to fibrinogen concentrations might be explained by other components of the fibrin gel whose concentrations were not considered but are known to affect gel properties potentially affecting cell behavior Calcium and chloride contained in the thrombin component for example are both known to influence fiber thickness and gel porosity 11 12 13 FXIII on the other hand can affect mechanical properties 14 and cell behavior as high concentrations inhibited process extension of human umbilical vein endothelial cells 15The objective of the present study was to modulate the mechanical and biological properties of fibrin keeping fibrinogen and thrombin constant To do so thrombin was diluted in buffers containing different calcium chloride and FXIII concentrations The relative structure of the resulting hydrogels was then estimated by light transmission measurements as it has been demonstrated that opacity of fibrin is directly related to its structure fiber mass/length ratio 16 Two buffer compositions leading to opaque thick fibers large pores 12 16 17 and translucent thin fibers narrow pores 12 16 17 gels were then selected For these gels mechanical properties were assessed by relaxation tests and biological properties were tested with short and longterm cell cultures For shortterm experiments both scaffolds were seeded either with mouse mesenchymal cell line C3H10T1/2 or with primary bovine bone marrowderived MSCs BMSCs and cultured up to 10 days At this time cell number morphology and distribution were evaluated For longterm experiments both translucent and opaque gels were seeded with BMSCs and cultured up to 17 and 24 days Cell morphology and distribution were then determined

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