In yet another published study, the MERLN researchers show the further optimization of vascular grafts by describing the fabrication of tubular scaffolds with different fiber alignments, which have robust mechanical properties and are leak-free and suturable. The scientists also studied the effect of these properties on the cell behavior of hMSC differentiated toward SMC-like cells. Endothelial cells were also seeded to obtain a construct comprising the intima and media layers of the artery.
The aim of this second MERLN study was to further develop the spinning technique that is able to align smooth muscle cells in a small-diameter vascular graft. In addition, simplified mathematical equations were formulated, based on the obtained results to predict the fiber diameter, distance between fibers and deposition angle. Primary smooth muscle cells were cultured on the scaffolds to assess whether the cells would morphologically mimic the tunica media of an artery. Finally, a co-culture with endothelial cells was done to assess whether the aligned primary smooth muscle cells influenced the morphology of the endothelial cells.
Smooth muscle cells play a pivotal role in maintaining blood pressure and remodeling of the extracellular matrix. These cells have a characteristic spindle shape and are aligned in the radial direction to aid in the constriction of any artery. Tissue-engineered grafts have the potential to recreate this alignment and offer a viable alternative to non-resorbable or autologous grafts. Specifically, with melt-spinning small-diameter fibers can be created that can align circumferentially on the scaffolds. In this study, a set of simplified equations were formulated to predict the final fiber parameters.
Effect of printhead speed on the scaffold fiber architecture. (A) Schematics showing the procedure used to quantify the angle between fibers. (B) Scanning electron microscopy images of produced scaffolds. (C) Quantification of angle between fibers from SEM images (n = 15–25, **** p ≤ 0.0001).
Melt-spinning for strength
Current additive manufacturing (AM) systems do not commonly implement the use of a rotational axis, which limits their application for the fabrication of tubular scaffolds. In a new study from the Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative Medicine, at Maastricht University and the Advanced Polymer Materials Group, University Politehnica of Bucharest, researchers developed a four-axis extrusion-based AM system similar to fused deposition modeling (FDM) has been developed to create tubular hollow scaffolds. The new additive process was dubbed “melt-spinning”.
Many tubular tissues such as blood vessels and trachea can suffer long-segmental defects through trauma and disease. With current limitations in the use of autologous grafts, the need for a synthetic substitute is of continuous interest as a possible alternative. Fabrication of these tubular organs is commonly done with techniques such as electrospinning and melt electrowetting using a rotational collector. Now a new method called melt-spinning and developed by MERLN researchers, based on a rotating 4-axis extrusion AM system could prove more efficient.
To test the ability of the scaffolds to hold liquids, Luer lock barbed plugs were introduced at each end of the scaffolds and connected to a syringe mounted on a syringe pump (shown in the figure below). Water with blue food coloring or a 5% BSA solution in PBS with red coloring flowed through the scaffolds. The solution with BSA was used as a “proxy” of blood. No leakage was observed at any of the flow rates tested (1.2, 10.2, and 25 mL/min). Despite the scaffolds being able to hold liquid, they presented high porosity (Supplementary Figure 3). Noteworthily, the scaffolds tested in the leak experiments had been previously used for the three-point bending test, indicating that the high strains applied had not negatively affected the scaffolds.
Smooth muscle cell alignment was monitored on the fabricated scaffolds. Finally, a co-culture of smooth muscle cells in direct contact with endothelial cells was performed to assess the influence of the smooth muscle cell alignment on the morphology of the endothelial cells. The results show that the equations were able to accurately predict the fiber diameter, distance and angle. Primary vascular smooth muscle cells aligned according to the fiber direction mimicking the native orientation. The co-culture with endothelial cells showed that the aligned smooth muscle cells did not have an influence on the morphology of the endothelial cells. In conclusion, we formulated a series of equations that can predict the fiber parameters during melt-spinning. Furthermore, the method described here can create a vascular graft with smooth muscle cells aligned circumferentially that morphologically mimics the native orientation.
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The implementation of a fourth axis in melt-spinning allows the possibility to create more complex tubular scaffold designs, as also described in the field of prosthetic implants. Other groups have implemented a fourth axis in their FDM system for biomedical applications, however often without communication between the rotational axis and the main system, which results in the creation of only helical designs. In addition to that, tubular scaffolds are generally not completely characterized by their mechanical properties and behavior for axial, radial and bending deformations.
A rectangular and a diamond pore design were further investigated for mechanical characterization, as a standard and a biomimicry pore geometry respectively. Three-point bending analyses revealed that the diamond pore design is more resistant to luminal collapse compared to the rectangular design. This data showed that by changing the scaffold pore design, a wide range of mechanical properties could be obtained. Furthermore, full control over scaffold design and geometry can be achieved with the developed 4-axis extrusion-based system, which has not been reported with other techniques. This flexibility allows the manufacturing of scaffolds for diverse tubular tissue regeneration applications by designing suitable deposition patterns to match their mechanical prerequisites.
Without the need of an electric field, the controllable and uncontrollable parameters of the manufacturing process are no longer a limitation. Consequently, it could be summed up in simplified equations to predict the fiber parameters in terms of fiber dimensions, angle of the fibers and resulting spacing. This could result in a more reproducible barrier layer. Much like SES, these microfibers require mechanical support. A four-axis melt extrusion-based system could provide this support by creating larger macro fibers around the smaller microfibers.
In a follow-up study, the MERLN researchers used the same melt-spinning technology to fabricate a mimetic vascular graft with tailorable fiber parameters. Melt-spinning allows the possibility of creating thin fibers around a mandrel without the need for an electric field by melting a polymer and depositing it directly on a spinning mandrel. The resulting fibers are highly aligned and reproducible with little variations in fiber dimensions. Recent advances in the technique led to the fabrication of core-shell fibers with enhanced mechanical properties.
A three-point bending test was performed to assess the mechanical behavior of the produced scaffolds (see figure on the left hand side). The 1° scaffolds showed a brittle behavior because they already broke at 10% strain. The 20° scaffolds were more robust, displaying no visible defects up to 60% strain, but started presenting clear openings of the fibers after that. The 30° scaffolds had no visible defects at the highest tested strain of 160%. The 20 and 30° scaffolds could be bent up to 160% strain (maximum tested) without kinking or scaffold breakage.
AM techniques, such as fused deposition modeling (FDM), bioplotting and other extrusion-based technologies, are suitable for increasing the mechanical integrity of a scaffold by manufacturing fibers with hundreds of micrometers. The main limitation in creating a tubular construct with current extrusion-based setups is the designs and geometries that can be manufactured, since for these overhanging and hollow structures a support material is required. This limitation is mainly caused by the fact that most systems use a layer-by-layer fabrication approach by subsequently depositing fibers to form an infill of each layer within the object contour and this process is repeated until the full 3D object is obtained. This deposition normally occurs in a flat substrate that moves in relation to the printhead in the X-Y plane and Z direction.
The aim of this study from MERLN was to show an extrusion-based AM technique similar to FDM with a synchronized fourth rotational axis that allows the fabrication of complex tubular geometries. In addition, the role of geometry in scaffold design and its influence on mechanical properties, such as radial compression, tensile strength and three-point bending was further studied.
Flexing rotational muscles
(A) Close-up of the leak test running at 25 mL/min. (B) Scaffold sutured onto the porcine carotid using a continuous running suture (6-0 Prolene).
Three-point bending test. (A) Macroscopic imaging at the beginning, at 20% strain, at 160% strain, and post-test. (B) Slope values from force–displacement measurements. (C) Maximum force values from force–displacement measurements. Two shades of gray indicate two separate experiments with a minimum n = 4 (total n = 10) (** p ≤ 0.01, *** p ≤ 0.001, n.d. = not determined).
The melt-spinning fabrication method from MERLN allowed fine control over fiber orientation in the circumferential direction (as shown in the figure above). The angle between fibers was quantified from images obtained by SEM. When the printhead was moved at a speed of 1 mm/s, the fibers were deposited parallel to each other (0 angle). At a speed of 10 mm/s, the angle between two fibers was 10.4 ± 1.0°; at a speed of 20 mm/s, the fiber angle was 19.3 ± 2.5°; and at a speed of 30 mm/s, the fiber angle was 33.6 ± 5.8°. The fiber diameter was 31.6 ± 3.9 μm, and it was not affected by the printhead speed. Fiber diameter can also be modified by changing the rotational speed of the collector. In this final MERLN study, this parameter was kept constant at 1060 rpm. For further testing, three fiber alignments were chosen (0, 19, and 33°) and, for easier labeling, were called 1, 20, and 30°.
