In Vitro Evaluation of PCL and P(3HB) as Coating Materials for Selective Laser Melted Porous Titanium Implants

Abstract

Titanium is widely used as a bone implant material due to its biocompatibility and high resilience. Since its Young's modulus differs from bone tissue, the resulting "stress shielding" could lead to scaffold loosening. However, by using a scaffold-shaped geometry, the Young's modulus can be adjusted. Also, a porous geometry enables vascularisation and bone ingrowth inside the implant itself. Additionally, growth factors can improve these effects. In order to create a deposit and release system for these factors, the titanium scaffolds could be coated with degradable polymers. Therefore, in the present study, synthetic poly-ε-caprolactone (PCL) and the biopolymer poly(3-hydroxybutyrate) (P(3HB)) were tested for coating efficiency, cell adhesion, and biocompatibility to find a suitable coating material. The underlying scaffold was created from titanium by Selective Laser Melting (SLM) and coated with PCL or P(3HB) via dip coating. To test the biocompatibility, Live Cell Imaging (LCI) as well as vitality and proliferation assays were performed. In addition, cell adhesion forces were detected via Single Cell Force Spectroscopy, while the coating efficiency was observed using environmental scanning electron microscopy (ESEM) and energy-dispersive X-ray (EDX) analyses. Regarding the coating efficiency, PCL showed higher values in comparison to P(3HB). Vitality assays revealed decent vitality values for both polymers, while values for PCL were significantly lower than those for blank titanium. No significant differences could be observed between PCL and P(3HB) in proliferation and cell adhesion studies. Although LCI observations revealed decreasing values in cell number and populated area over time on both polymer-coated scaffolds, these outcomes could be explained by the possibility of coating diluent residues accumulating in the culture medium. Overall, both polymers fulfill the requirements regarding biocompatibility. Nonetheless, since only PCL coating ensured the maintenance of the porous implant structure, it is preferable to be used as a coating material for creating a deposit and release system for growth factors.

Keywords: osteoblast; poly(3-hydroxybutyrate); polycaprolactone; titanium scaffold.

Figure 1

Schematic side view (A) top view (B) and 3D view of the scaffold geometry; (C) The dotted line marks the division of the implant into an external and internal area.

Figure 2

Exemplary micrograph image of a poly-ε-caprolactone (PCL)-coated titanium scaffold after cross-section preparation (black = titanium with pores; white rims = PCL coating; red = embedding medium; scale bar = 44.05 µm).

Figure 3

Experimental set-up for vitality and proliferation assays on PCL and poly(3-hydroxybutyrate) (P(3HB)) foils.

Figure 4

Experimental set-up for vitality and proliferation assays on titanium scaffolds.

Figure 5

Force-displacement (FD) curve of a single osteoblast attached to PCL-coated dish surface for 180 s. The curve shows the maximum detachment force (red circle) of the cell and single cell-PCL surface bindings that were separated by moving the cell away from the PCL surface (small force steps, arrow).

Figure 6

Representative environmental scanning electron microscopy (ESEM) micrographs of uncoated (A,D), PCL-coated (B,E) and P(3HB)-coated (C,F) porous titanium scaffolds in overview (A–C; scale bar = 1 mm) and in detail (D–F; scale bar = 50 µm (uncoated), 100 µm (PCL), 200 µm (P(3HB)). Within the red circles cavities between microparticles are shown on the uncoated titanium scaffolds, whereas the blue asterisks mark uncoated titanium after applying P(3HB) coating.

Figure 7

Representative ESEM micrographs of PCL ((A,B) first side and (A’,B’) second side) and P(3HB) ((C,C’) fist side and (D,D’) second side) foils in overview ((A,A’) and (C,C’); 100-fold magnification, scale bar: 200 µm) and in detail ((B,B’) and (D,D’); 1000-fold magnification, scale bar: 20 µm).

Figure 8

Vitality of green fluorescent protein (GFP)-osteoblasts on the blank well bottom (positive control), PCL and P(3HB) foils, as well as on titanium scaffolds (A). The Kruskall–Wallis Test followed by the Wilcoxon’s rank-sum test showed a significantly lower cell vitality on both PCL and P(3HB) compared to the well bottom. Also, significantly lower cell vitality on PCL compared to the titanium scaffolds was revealed. Proliferation index of GFP-osteoblasts on the blank well bottom (positive control), PCL and P(3HB) foils, as well as on titanium scaffolds (B). No significant difference between the materials could be found using the Kruskall–Wallis Test (horizontal line = median, whiskers = minimal and maximal value, circle = outlier, * = p < 0.05, *** = p < 0.001).

Figure 9

GFP-linked osteoblasts on an uncoated (A–C), PCL-coated (D–F) or P(3HB)-coated (G–I) titanium scaffold on day 0 (A,D,G), 3 (B,E,H) and 7 (C,F,I). The pictures were taken at a 10-fold magnification (scale bar: 250 µm).

Figure 10

Number of GFP-osteoblasts on uncoated, PCL-coated and P(3HB)-coated titanium scaffolds in the course of 7 days (A), as well as the populated area of these implants (B) and the cell spreading area of the osteoblasts (C) in the same temporal dimensions. Using the F-test for interaction between time and materials, significant differences could be revealed between the populated area of uncoated and both PCL- and P(3HB)-coated scaffolds. Also, significant differences could be found for the cell spreading area of uncoated scaffolds compared to coated ones (circles = single values for uncoated titanium scaffolds, triangles = single values for PCL-coated scaffolds, rhombi = single values for P(3HB)-coated scaffolds, lines = regression curves, * = p < 0.05, ** = p < 0.01).

Figure 11

Number of GFP-osteoblasts on uncoated, PCL-coated and P(3HB)-coated titanium scaffolds in the course of 7 days (A), as well as the populated area of these implants (B) and the cell spreading area of the osteoblasts (C) in the same temporal dimensions. Using the F-test for interaction between time and materials significant differences could be revealed between the populated area of uncoated and both PCL- and P(3HB)-coated scaffolds. Also, significant differences could be found for the cell spreading area of uncoated scaffolds compared to coated ones (circles = single values for uncoated titanium scaffolds, triangles = single values for PCL coated scaffolds, rhombi = single values for P(3HB)-coated scaffolds, lines = regression curves).