Fig 1.
Schematic overview of the methodology.
The poly-ε-lysine and hyaluronic acid (PEL-10/HA144-1) coating is sprayed on titanium (Ti) and ultra-high-molecular-weight-polyethylene (UHMWPE). Coating characterization, antibacterial properties, and biocompatibility were assessed in parallel. All bacteria are American Type Culture Collection (ATCC) strains. Mouse fibroblasts (L929, ATCC cell line CCL 1, NCTC clone 929) and human osteoblast-like cells (Saos-2, CLS, CLS 300331, epithelial-like) were used for biocompatibility assessment.
Fig 2.
PEL-10/HA144-1 coating visualization.
PEL-10/HA144-1 coating labeled with Poly-L-Lysine coupled with fluorescein isothiocyanate (PLL-FITC) (green) was observed with confocal microscopy on both titanium (Ti) and ultra-high-molecular-weight-polyethylene (UHMWPE) samples (A). Scanning Electron Microscopy (SEM) images of Ti samples without (left) and with (right) PEL-10/HA144-1 coating (B). SEM settings: 10.0kV, WD10mm, PC 40.0, 3000x.
Fig 3.
The water contact angle (WCA).
Visualization of the WCA of titanium and UHMWPE substrates, either uncoated or coated with PEL-10/HA144-1 (A). The mean of the WCA of three locations on three samples is displayed ±SD (B). Statistical differences found with an unpaired t-test are presented with ****P < 0.0001.
Table 1.
Antibacterial activity R and reduction in CFU (%). Induced by the PEL-10/HA144-1 coating in the antibacterial activity test.
Fig 4.
Results from the antibacterial activity test.
After 24h of incubation with S. aureus (A) and E. coli (B), viable bacteria were collected from the samples. The starting inoculum is indicated by a red dotted bar. Green bars represent Titanium samples, and green/black striped bars represent UHMWPE samples. Each bar represents the average ± SD of three experiments, each including three samples. Statistical differences found with an unpaired t-test are presented with ** p < 0.01, and ****P < 0.0001. Each bar represents the average ± SD of three experiments, each including three samples. Statistical differences found with an unpaired Mann-Whitney U-test are presented with ****P < 0.0001.
Table 2.
Log10 and percentage reductions between the PEL-10/HA144-1 coated samples and the uncoated samples in the bacterial adhesion test.
Fig 5.
Results from the bacterial adhesion test.
Viable bacteria, S. aureus (A and B) or E. coli (C and D), that adhered to the samples after 4h (A and C) or 24h (B and D) of incubation, were collected from the samples. The starting inoculum was 2.23 × 107 CFU/mL and 3.28 × 107 CFU/mL for S. aureus ATCC25923 and E. coli ATCC25922, respectively. Green bars represent titanium samples, and green/black striped bars represent UHMWPE samples. Each bar represents the average ± SD of three experiments, each including three samples. Statistical differences found with an unpaired t-test are presented with ****P < 0.0001. Statistical differences found with an unpaired t-test are presented with ■■■ = 0.0002 and ■■■■ < 0.0001. Statistical differences found with a Mann-Whitney U-test are presented with ****P < 0.0001.
Fig 6.
Scanning Electron Microscopy images.
Titanium (Ti) samples after 24h incubation in a bacterial adhesion test with S. aureus (A and B), and E. coli (C and D), without (A and C) and with (B and D) PEL-10/HA144-1 coating. Settings: 10.0 kV, WD 10 mm, PC 40.0, 3000x zoom. C and E) arrows indicate examples of accumulation of bacteria and biofilm structures; D and F) arrows indicate examples of single S. aureus and E. coli bacteria that are left.
Fig 7.
As found by the WST-1 assay for the L929 cells (A) and Saos-2 cells (C), and corresponding cytotoxicity as found by the LDH assay (B for L929 and D for Saos-2 cells). Green bars represent Titanium samples, and green/black striped bars represent UHMWPE samples. Each bar represents the average ± SD of three experiments, each including three samples. Significant differences as found by the Kruskal-Wallis multiple comparison test are displayed in (A) and (C) for untreated cells versus all other samples, and in all graphs for the uncoated sample extract versus all percentages of the coated samples. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 8.
Fluorescent staining of nucleus (DAPI) and actin (Phalloidin) in Saos-2 osteoblast-like cells (A).
Cells have been cultured for 24 h on polystyrene control, uncoated Ti, and coated Ti. Standard morphology is observed in the control group, where the morphology of the cells on the uncoated Ti is slightly different. The morphology of the cells on the coated Ti is distorted compared to the control. Fluorescent images (10x magnification) were analysed in CellProfiler to quantify the nucleus (B) and cytoskeleton (C) differences between conditions. The area was calculated by identifying objects in the pictures, their size, and the number of pixels. The spread of data points is large due to the difference in the size of the cells. Significant differences can be observed between all conditions for the nucleus and cytoskeleton area, as can be observed in Fig 8A. Data represents one experiment with three technical replicates. ****p < 0.0001. Whiskers and outliers are plotted with the Tukey method.
Fig 9.
Cell viability of Saos-2 osteoblast-like cells assessed by direct contact.
With coated and uncoated Ti discs, the control is a polystyrene well plate. A) LDH measurements at day 1, where LDH max was treated with 1% Triton X. B) Protein quantification over 14 days. The first week shows no statistical difference between Ti and Ti coating. Additionally, the µg proteins only increased after day 7. At day 14, a statistical difference between Ti and Ti coating can be observed. C) Metabolic activity normalized to protein content on day 1, 3, and 7. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 10.
The osteoblast phenotype of Saos-2 cells, assessed using mineralization and secretion of Osteoprotegerin.
A) A significant difference is observed in the secreted Osteoprotegerin per cell between control and stimulated conditions for the coated Ti. B) The mineralization assay shows that cells can mineralize in the presence of the coating while stimulated with osteogenic medium. The stimulated conditions are osteogenic medium supplemented with 1α,25-dihydroxyvitamin D3. *p < 0.05.