Electrospun Poly-L-Lactic Acid Scaffolds Surface-Modified via Reactive Magnetron Sputtering Using Different Mixing Ratios of Nitrogen and Xenon

Abstract

Controlled regeneration processes involving tissue growth using the surface and structure of scaffolds, are actively used in tissue engineering. Reactive magnetron sputtering is a versatile surface modification method of both metal and polymer substrates, as the properties of the formed coatings can be modified in a wide range by changing the process parameters. In magnetron sputtering, the working gas and its composition have an influence on the chemical composition and physical characteristics of the obtained coatings. However, there are no studies addressing the influence of the nitrogen/xenon gas mixture ratio in direct current magnetron sputtering on the deposition rate, physicochemical and in vitro properties of surface-modified biocompatible poly-L-lactic acid scaffolds. In this study, the application of mixtures of nitrogen and xenon in various ratios is demonstrated to modify the surface of non-woven poly-L-lactic acid scaffolds by direct current magnetron sputtering of a titanium target. It has been found that the magnetron sputtering parameters chosen do not negatively influence the morphology of the prepared scaffolds, but increase the hydrophilicity. Moreover, quantitative spectroscopic analysis results indicate that the formed coatings are primarily composed of titanium oxide and titanium oxynitride compounds and is dependent on the gas mixture ratio only to a certain extent. Atomic force microscopy investigations of the roughness of the fibers of the electrospun scaffolds and the thickness of the coatings formed on them show that the considerable variations observed in the intrinsic fiber reliefs are due to the formation of a fine layer on the fiber surfaces. The observed decrease in roughness after plasma modification is due to temperature and radiation effects of the plasma. In vitro experiments with human osteosarcoma cells show that the scaffolds investigated here have no cytotoxic effect on these cells. The cells adhere and proliferate well on each of the surface-modified electrospun scaffolds, with stimulation of cell differentiation in the osteogenic direction.

Keywords: HOS cells; electrospun PLLA scaffold; nitrogen-containing titanium coating; proliferative activity; reactive magnetron sputtering; working gas mixtures.

Figure 1

Experimental scheme of the fabrication of PLLA scaffolds, their surface modification via reactive DC magnetron sputtering of a titanium target using a gas mixture of nitrogen and xenon at different mixing ratios, and the investigation methods applied in this study.

Figure 2

Scanning electron microscope (SEM) micrographs on the left, histograms of the fiber diameters of the scaffold samples in the middle, and histograms of the pore areas of the scaffolds on the right: (a) Control samples (PLLA scaffolds without surface modification), (b) N₂ 100%, (c) N₂ 75% + Xe 25%, (d) N₂ 50% + Xe 50%, (e) N₂ 25% + Xe 75%, (f) Xe 100%.

Figure 3

(a) Glycerol contact angle (GCA) for the PLLA scaffold samples under investigation, and (b) coating thicknesses of all surface-modified samples.

Figure 4

High-resolution spectra obtained via XPS for all PLLA scaffold samples that are surface-modified with a mixture of nitrogen and xenon at mixing ratios: (a) Ti2p, (b) O1s, (c) N1s and (d) C1s.

Figure 5

Root mean square (RMS) roughness obtained by atomic force microscopy (AFM) at two different sized scanning areas of unmodified and surface-modified PLLA scaffold samples obtained at different mixing ratios of the working gases nitrogen and xenon at (a) 40 × 40 μm² scanning area and (b) 3 × 3 μm² scanning area.

Figure 6

(a) Amount for HOS cells present at the PLLA scaffold sample surfaces surface-modified using different mixing ratios of the working gases nitrogen and xenon. (b) Proliferative activity of HOS cells assessed via MTT assay (measured by absorbance and presents therefore the optical density).

Figure 7

Expression of (a) BGLAP and (b) SPP1 genes in HOS cells at day 14 after the start of incubation. Here, the P-Control samples indicate the level of proliferative activity of HOS cells placed into a polymeric Petri dish and then set to 100%. The statistical legend is as follows: *—statistically significant differences to the control samples are p < 0.05; #—the statistically significant differences to the samples N₂ 75% + Xe 25% are at p < 0.05; α—the statistically significant differences to the samples Xe 100% being p < 0.05.