(I-345) Mechanical Characterization of 3D Printed Fibrin Microthreads

Introduction:Fibrin, the fundamental provisional matrix protein during wound healing, plays a major role in tissue regeneration by providing chemical and topological cues for cell adhesion and infiltration. During the early stage of wound healing, native fibrin scaffolds degrade over time and are replaced by newly synthesized extracellular matrix. Fibrin can be easily isolated from the patient’s own blood, which makes it a great candidate for personalized medicine applications, but its use as a bioink is somewhat limited by its high viscosity [1]. We hypothesized that cell inclusion and an increase of fibrinogen concentration increase the viscosity of the bioink, resulting in different mechanical properties. Here we investigated the uniaxial tensile properties of 3D printed fibrin threads as a function of fibrinogen concentration, processing methods, and in vitro cell culture.


Materials and Methods:Bovine fibrinogen was dissolved in HEPES buffered saline (20 mM HEPES, 0.9% w/v NaCl in DI-H2O) to a concentration of 10 mg, 30 mg, 50 mg, or 70 mg of fibrinogen/ml. Bovine thrombin was initially diluted in HEPES buffered saline (40U/ml) and then further diluted 1.5:8.5 with a 40mM solution of calcium chloride in DI-H2O. To create fibrin microthreads, the fibrinogen and thrombin solutions were loaded in a custom device and printed through a 32-cm long segment of polyethylene tubing (0.86 mm inner diameter) at 0.255 ml/m in a HEPES bath (pH 7). After allowing the threads to polymerize for 30 minutes, they were statically stretched 1.5- or 2-fold to investigate strain hardening. 10-mg fibrinogen aliquots were also used to print threads loaded with C2C12 mouse myoblasts (4x10^6 cells/ml). In this case, HEPES buffer was substituted with cell media. The threads were kept in proliferation medium (50:50 High glucose DMEM/F-12, 10% fetal bovine serum, 1% penicillin/streptomycin, 1% amphotericin B) for 3 days and were then transferred to myoblast differentiation medium (50:50 High Glucose DMEM/F-12, 1x insulin-transferrin-selenium, 2% horse/equine serum, 1% penicillin/streptomycin, 1% amphotericin B, 100 μg/ml ascorbic acid) for 4 days. Uniaxial tensile mechanical behavior was investigated at 50% strain rate using an ElectroPuls E1000 (Instron, Norwood, MA). Tests were run until failure. Tangential moduli were calculated from the averaged stress – strain curve of each group (n = 5). Statistical significance was determined via one-way ANOVA using SigmaPlot 14.0 (Systat Software Inc.) and p < 0.05.


Results, Conclusions, and Discussions:The main obstacle to the adoption of fibrin as a bioink for 3D printing is its high viscosity, which we expected to affect the mechanical properties of printed constructs. We investigated the effects of cell inclusion and fibrinogen concentration on the mechanical properties of 3D printed microthreads. All groups showed a lower tangential modulus than scar tissue by several orders of magnitude (10^5 MPa vs < 1 MPa). The elastic modulus of the microthreads printed with higher fibrinogen concentration ( > 50 mg/ml) could be increased by statically stretching the threads to induce strain hardening after the extrusion. However, this phenomenon was observed only in threads stretched to 2 times their original length. All threads maintained their mechanical properties up to 20% strain, which is a range sufficient for most tissue engineering applications. Even in their unstretched state, the threads recapitulated the tangential modulus of load-bearing soft tissues (e.g. human heart ventricle 0.01 - 0.4 MPa) more closely than collagen. This suggests that they can be integrated in hydrogel-based scaffolds to control anisotropy, for applications such as myocardial or skeletal muscle regeneration. To study the effect of cell inclusion on the mechanical properties of printed threads, we suspended murine myoblasts in the fibrinogen solution with a final concentration (5 mg/ml) comparable to that occurring in the blood of healthy individuals (2-4 mg/ml). We found no significant difference in terms of elastic modulus. Despite the addition of ascorbic acid, in vitro cell culture and myoblasts differentiation did not degrade the fibrin threads over the span of 7 days. No difference was found between the acellular threads extruded in cell culture medium and those extruded in the HEPES bath. While our results suggest that fibrin threads are a suitable platform for a variety of tissue engineering applications, more studies are warranted to investigate the influence of different cell types, higher cell density, and in vivo fibrinolysis on long term stability and mechanical properties of fibrin scaffolds.


Acknowledgements: This study was funded by the American Heart Association Institutional Research Enhancement Award 953260.


References: [1] de Melo, B. A. G., Jodat, Y. A., Cruz, E. M., Benincasa, J. C., Shin, S. R., & Porcionatto, M. A. (2020). Strategies to use fibrinogen as bioink for 3D bioprinting fibrin-based soft and hard tissues. Acta biomaterialia, 117, 60–76