Melt electrowriting of PLA, PCL, and composite PLA/PCL scaffolds for tissue engineering application

Abstract

Fabrication of well-ordered and bio-mimetic scaffolds is one of the most important research lines in tissue engineering. Different techniques have been utilized to achieve this goal, however, each method has its own disadvantages. Recently, melt electrowriting (MEW) as a technique for fabrication of well-organized scaffolds has attracted the researchers' attention due to simultaneous use of principles of additive manufacturing and electrohydrodynamic phenomena. In previous research studies, polycaprolactone (PCL) has been mostly used in MEW process. PCL is a biocompatible polymer with characteristics that make it easy to fabricate well-arranged structures using MEW device. However, the mechanical properties of PCL are not favorable for applications like bone tissue engineering. Furthermore, it is of vital importance to demonstrate the capability of MEW technique for processing a broad range of polymers. To address aforementioned problems, in this study, three ten-layered box-structured well-ordered scaffolds, including neat PLA, neat PCL, and PLA/PCL composite are fabricated using an MEW device. Printing of the composite PLA/PCL scaffold using the MEW device is conducted in this study for the first time. The MEW device used in this study is a commercial fused deposition modeling (FDM) 3D printer which with some changes in its setup and configuration becomes prepared for being used as an MEW device. Since in most of previous studies, a setup has been designed and built for MEW process, the use of the FDM device can be considered as one of the novelties of this research. The printing parameters are adjusted in a way that scaffolds with nearly equal pore sizes in the range of 140 µm to 150 µm are fabricated. However, PCL fibers are mostly narrower (diameters in the range of 5 µm to 15 µm) than PLA fibers with diameters between 15 and 25 µm. Unlike the MEW process of PCL, accurate positioning of PLA fibers is difficult which can be due to higher viscosity of PLA melt compared to PCL melt. The printed composite PLA/PCL scaffold possesses a well-ordered box structure with improved mechanical properties and cell-scaffold interactions compared to both neat PLA and PCL scaffolds. Besides, the composite scaffold exhibits a higher swelling ratio than the neat PCL scaffold which can be related to the presence of less hydrophobic PLA fibers. This scaffold demonstrates an anisotropic behavior during uniaxial tensile test in which its Young's modulus, ultimate tensile stress, and strain to failure all depend on the direction of the applied tensile force. This anisotropy makes the composite PLA/PCL scaffold an exciting candidate for applications in heart tissue engineering. The results of in-vitro cell viability test using L929 mouse murine fibroblast and human umbilical vein endothelial (HUVEC) cells demonstrate that all of the printed scaffolds are biocompatible. In particular, the composite scaffold presents the highest cell viability value among the fabricated scaffolds. All in all, the composite PLA/PCL scaffold shows that it can be a promising substitution for neat PCL scaffold used in previous MEW studies.

Figure 1

Schematic of the experimental setup (drawn on the BioRender website (https://biorender.com) by the authors).

Figure 2

FDM 3D printer used in this study as the MEW device.

Figure 3

Print head and heating section of the modified FDM device and the configuration used to confine the high voltage to nozzle head and heating box.

Figure 4

SEM images of the melt elecrowritten scaffolds, (a–c) well-ordered box structure of the neat PCL, neat PLA, and composite PLA/PCL scaffolds, respectively, (d) bonding and connection of two fibers (the below one is PLA and the upper one is PCL) melt electrowritten in two consecutive layers perpendicularly on each other, and (e,f) surface of one of the PCL and PLA fibers, respectively.

Figure 5

Stress vs. strain curve for 3 scaffolds (the test is conducted in two directions for the composite PLA/PCL scaffold, one is the direction of printing the PLA fibers and the other is the direction of printing the PCL fibers which is perpendicular to the first direction) (number of samples for each measurement = 3) (Statistical analysis with one-way ANOVA shows statistical significance with p-values less than 0.05).

Figure 6

In vitro cytotoxicity and cell viability of the scaffolds, (a,b) biocompatibility of the scaffolds for L929 and HUVEC cells obtained by OD measurements, respectively, and (c,d) cell viability of the scaffolds for L929 and HUVEC cells, respectively (number of samples for each measurement = 3) (for OD charts: *shows p < 0.05, **demonstrates p < 0.01, and ***represents p < 0.001) (for cell viability charts: statistical significance is shown with negligible p value).

Figure 7

(a–c) L929 cells adhesion to the surface of the neat PCL, neat PLA, and composite PLA/PCL scaffolds, respectively, and (d–f) attachment of HUVEC cells to the surface of the neat PCL, neat PLA, and composite PLA/PCL scaffolds, respectively.

Figure 8

(a–c) LIVE/DEAD staining results of L929 cells for the neat PCL, neat PLA, and composite PLA/PCL scaffolds, respectively, (d–f) LIVE/DEAD staining results of HUVEC cells for the neat PCL, neat PLA, and composite PLA/PCL scaffolds, respectively, and (g) view of HUVEC cells on the crossing fibers of the composite PLA/PCL scaffold showing the large number of alive cells on the intersection.