Undergraduate Student Massachusetts Institute of Technology Neponsit, New York, United States
Introduction:: Three-dimensional bioprinting is an emerging fabrication technique borrowing insights from mechanical engineering and biology. Fibrin gel is commonly used in tissue engineering as a scaffold in 3D cell culture since it is biocompatible and has tunable mechanical properties. The biggest challenge with using fibrin as a material in three-dimensional bioprinting is a lack of shape fidelity to its intended computer aided design [1]. Two important parameters in a type of 3D bioprinting known as “freeform reversible embedding of suspended hydrogels” are the starting height of a print within its support bath and the speed at which the ink is extruded from the nozzle. Establishing the dependance of shape fidelity and stiffness on these parameters will set the foundation for creating complex fibrin scaffolds for tissue growth.
Materials and Methods:: To study the effect that extrusion speed and starting depth has on shape fidelity and stiffness, fibrin rectangles were printed from the FlashForge 3D printer. This printer was designed for fused deposition modeling but was converted into a bioprinter that uses a syringe extruder, as shown in Figure 1. Fibrin “ink” was made from fibrinogen at 4mg/mL and cell growth media and loaded into the syringe. The low viscosity ink could not be printed independently, so a support bath was made from a blended gelatin solution, which will be referred to as a “gelatin slurry.” The support bath was supplemented with thrombin at 0.15U/mL as a crosslinking agent. Rectangular prisms measuring 1cm x 2mm x 2mm were printed in individual petri dishes filled with the gelatin slurry. The samples were placed in a 37C incubator for 30 minutes after printing to melt the gelatin slurry and removed and stored in cell growth media at 4C. Images were taken of the fibrin prints in cell media, and using MATLAB, the length, width, and area of the samples were determined, as shown in Figure 2. Following 2 days at 4C, the samples were removed from the media and texture analyzed using a Stable Micro Systems Texture Analyzer with Exponent Software. This machine performed a compression test on the samples to reveal their stiffness through measuring the force necessary to compress them to 50% of their original height. This machine also provided data to determine the height of the samples. See Figure 1.
Results, Conclusions, and Discussions:: The aim of this study was to quantify how shape the fidelity of 3D bioprinted fibrin is affected by extrusion speed and starting height. Starting height did not have any significant effect on the dimensions of the print. The length, width, and height of the prints increased by 217%, 496%, and 58%, respectively, per every microliter per second increase in extrusion speed, as shown in Figure 3. Thus, print speed showed a significant direct relationship with all of the construct dimensions. The upward trend of print dimension with increasing speed is due to the greater volume of ink being extruded from the syringe nozzle per unit time.
The relationship between the extrusion factor and size of individual dimensions was not constant among length, width, and height. Thus, when creating a CAD model to print fibrin in the future, it is necessary to scale the length, width, and height dimensions differently to achieve the desired geometry post printing and processing.
The average elastic modulus of the samples printed with an extrusion factor of 0.5 ul/sec was 85.4 ± 24.8 kPa and the average elastic modulus of the samples printed using an extrusion factor of 0.75 ul/sec was 26.9 ± 7.7 kPa. See Figure 4. The modulus of human skeletal muscle is 24.7 ± 3.5kPa and the modulus of human cardiac muscle is 100.3 ± 10.7kPa, which are close in value to the average moduli of the 0.75 ul/sec and 0.5 ul/sec groups respectively [2].
The starting height of the print did not have a statistically significant impact on its elastic modulus. The variation in stiffness between samples printed with different extrusion speeds can be attributed to the size of the sample. The clotting agent in the support bath will have trouble fully penetrating larger volume prints, which is likely the cause of this modulation in stiffness between samples. Thus, if larger prints are executed, it may be necessary to increase the concentration of thrombin.
The next steps are to 3D bioprint muscle cells suspended in fibrinogen ink and study their growth and differentiation in the presence of magnetic stimulation.
Acknowledgements (Optional): :
References (Optional): : [1] de Melo, B. A. G., Jodat, Y. A., Cruz, E. M., Benincasa, J. C., Shin, S. R., and Porcionatto, M. A., 2020, “Strategies to Use Fibrinogen as Bioink for 3D Bioprinting Fibrin-Based Soft and Hard Tissues,” Acta Biomaterialia, 117, pp. 60–76.
[2] Ogneva, I. V., Lebedev, D. V., and Shenkman, B. S., 2010, “Transversal Stiffness and Young’s Modulus of Single Fibers from Rat Soleus Muscle Probed by Atomic Force Microscopy,” Biophys J, 98(3), pp. 418–424.
