The Mineralization of Various 3D-Printed PCL Composites

Abstract

In this project, different calcification methods for collagen and collagen coatings were compared in terms of their applicability for 3D printing and production of collagen-coated scaffolds. For this purpose, scaffolds were printed from polycaprolactone PCL using the EnvisionTec 3D Bioplotter and then coated with collagen. Four different coating methods were then applied: hydroxyapatite (HA) powder directly in the collagen coating, incubation in 10× SBF, coating with alkaline phosphatase (ALP), and coating with poly-L-aspartic acid. The results were compared by ESEM, µCT, TEM, and EDX. HA directly in the collagen solution resulted in a pH change and thus an increase in viscosity, leading to clumping on the scaffolds. As a function of incubation time in 10× SBF as well as in ALP, HA layer thickness increased, while no coating on the collagen layer was apparently observed with poly-L-aspartic acid. Only ultrathin sections and TEM with SuperEDX detected nano crystalline HA in the collagen layer. Exclusively the incubation in poly-L-aspartic acid led to HA crystals within the collagen coating compared to all other methods where the HA layers formed in different forms only at the collagen layer.

Keywords: 3D printing; PCL scaffolds; alkaline phosphatase; collagen coating; hydroxyapatite; poly-L aspartic acid.

Figure 1

Overview of immunostaining; (a): uncoated scaffolds; (b): collagen-coated scaffolds. Images taken with Olympus BX-53 Fluorescence microscope @ 500 ms illumination time.

Figure 2

ESEM images of collagen coating by incubation; the red area in (a) is shown enlarged in (b); the uncoated scaffold is shown in (c).

Figure 2

ESEM images of collagen coating by incubation; the red area in (a) is shown enlarged in (b); the uncoated scaffold is shown in (c).

Figure 3

Comparison of collagen-HA coatings: (a): gelled collagen-HA coating; (b): collagen-HA coating, which has been diluted with acetic acid.

Figure 4

Comparative ESEM images of the coatings formed by incubation in 10× SBF. Incubation time varies from top to bottom: 1, 2, 4, and 8 h. The left image always shows the scaffold at 188× magnification, and the right image shows the crystalline structures and agglomerates on the surface. Because the crystalline structures and agglomerates increase over the incubation time, the magnification factor also varies. ESEM images were taken with a FEI Quanta FEG 250 @ 20 kV, 130 Pa, Large Field Detector.

Figure 4

Comparative ESEM images of the coatings formed by incubation in 10× SBF. Incubation time varies from top to bottom: 1, 2, 4, and 8 h. The left image always shows the scaffold at 188× magnification, and the right image shows the crystalline structures and agglomerates on the surface. Because the crystalline structures and agglomerates increase over the incubation time, the magnification factor also varies. ESEM images were taken with a FEI Quanta FEG 250 @ 20 kV, 130 Pa, Large Field Detector.

Figure 5

Comparison of the ESEM images for the different incubation times in PIM; (left): fracture edge of a cylindrical strand with coating on the outer surface; (right): surface of a strand with agglomerations of the crystalline structures.

Figure 6

ESEM images of fracture edge (left) and surface of a strand (center), and magnification of the surface (right) of a sample treated with poly-L-aspartic acid.

Figure 7

MicroCT images of samples incubated in: (a): ALP-activated PIM; (b): poly-ASP; (c): uncoated sample.

Figure 8

TEM (a) and EDX (b) images of thin sections of collagen-coated PCL, post-treated with poly-L-aspartic acid; TEM image taken with TALOS 200X, 185,000× magnification, HFW 546 nm, STEM HAADF, EDX measuring with FEI SuperX EDX System.