3D Bioprinting of Novel Biocompatible Scaffolds for Endothelial Cell Repair

Abstract

The aim of this study was to develop and evaluate an optimized 3D bioprinting technology in order to fabricate novel scaffolds for the application of endothelial cell repair. Various biocompatible and biodegradable macroporous scaffolds (D = 10 mm) with interconnected pores (D = ~500 µm) were fabricated using a commercially available 3D bioprinter (r3bEL mini, SE3D, USA). The resolution of the printing layers was set at ~100 µm for all scaffolds. Various compositions of polylactic acid (PLA), polyethylene glycol (PEG) and pluronic F127 (F127) formulations were prepared and optimized to develop semi-solid viscous bioinks. Either dimethyloxalylglycine (DMOG) or erythroprotein (EPO) was used as a model drug and loaded in the viscous biocompatible ink formulations with a final concentration of 30% (w/w). The surface analysis of the bioinks via a spectroscopic analysis revealed a homogenous distribution of the forming materials throughout the surface, whereas SEM imaging of the scaffolds showed a smooth surface with homogenous macro-porous texture and precise pore size. The rheological and mechanical analyses showed optimum rheological and mechanical properties of each scaffold. As the drug, DMOG, is a HIF-1 inducer, its release from the scaffolds into PBS solution was measured indirectly using a bioassay for HIF-1α. This showed that the release of DMOG was sustained over 48 h. The release of DMOG was enough to cause a significant increase in HIF-1α levels in the bioassay, and when incubated with rat aortic endothelial cells (RAECs) for 2 h resulted in transcriptional activation of a HIF-1α target gene (VEGF). The optimum time for the increased expression of VEGF gene was approximately 30 min and was a 3-4-fold increase above baseline. This study provides a proof of concept, that a novel bioprinting platform can be exploited to develop biodegradable composite scaffolds for potential clinical applications in endothelial cell repair in cardiovascular disease (CVD), or in other conditions in which endothelial damage occurs.

Keywords: 3D bioprinting; DMOG; EPO; biocompatible; endothelial cell; polylactic acid; scaffolds.

Figure 1

Schematic diagram of (a) the optimized 3D bioprinting process, (b) the extrudable bioink, and (c) the printed scaffold with 3D texture.

Figure 2

The texture and morphology of the semi-solid bioinks under an optical microscope of formulations F1 (F127:drug 4:1), F2 (PLA/PEG:drug 49:1), F3 (F127:drug 1:0), and F4 (PLA/PEG: drug 49:0) (scale 10 microns).

Figure 3

Rheology data of the bioink formulations represented by storage modulus (G′) and loss modulus (G′′) both in temperature (test run at 5–50 °C) and frequency sweeps (test run at 25 °C) for formulation F127/EPO 4:1.

Figure 4

SEM images of (a) bioprinted, and (b) cross-section of PLA/PEG/EPO-based scaffolds.

Figure 5

Force and travel time (as function of strain) profiles of the bioink formulations: F1 (F127:drug 4:1), F2 (PLA/PEG:drug 49:1), F3 (F127:drug 1:0), and F4 (PLA/PEG: drug 49:0) prior to the 3D printing applications (n = 3) of porous scaffolds.

Figure 6

DSC thermal transitions of the blank polymeric formulations and the scaffolds.

Figure 7

The release of HIF-1α inducer; (a) DMOG from 3D printed scaffolds in phosphate buffered saline solution (PBS) at room temperature. DMOG was used at a concentration of 30% in all the matrices prepared. (The biomaterial mixture contained PLA/PEG in the ratio 70:0, 60:10 and 50:20), (b) Fold change in VEGF gene expression in cell lysates after treating rat aortic endothelial cells with different samples of DMOG released at different time points (0, 0.5 and 3 h), and VEGF gene expression VEGF mRNA levels were measured as arbitrary units versus the untreated samples. (c) release of EPO from the 3D printed scaffolds. Data are the mean ± SEM of 6 samples. P < 0.01.