Is Silver Addition to Scaffolds Based on Polycaprolactone Blended with Calcium Phosphates Able to Inhibit Candida albicans and Candida auris Adhesion and Biofilm Formation?

Abstract

Candida spp. periprosthetic joint infections are rare but difficult-to-treat events, with a slow onset, unspecific symptoms or signs, and a significant relapse risk. Treatment with antifungals meets with little success, whereas prosthesis removal improves the outcome. In fact, Candida spp. adhere to orthopedic devices and grow forming biofilms that contribute to the persistence of this infection and relapse, and there is insufficient evidence that the use of antifungals has additional benefits for anti-biofilm activity. To date, studies on the direct antifungal activity of silver against Candida spp. are still scanty. Additionally, polycaprolactone (PCL), either pure or blended with calcium phosphate, could be a good candidate for the design of 3D scaffolds as engineered bone graft substitutes. Thus, the present research aimed to assess the antifungal and anti-biofilm activity of PCL-based constructs by the addition of antimicrobials, for instance, silver, against C. albicans and C. auris. The appearance of an inhibition halo around silver-functionalized PCL scaffolds for both C. albicans and C. auris was revealed, and a significant decrease in both adherent and planktonic yeasts further demonstrated the release of Ag⁺ from the 3D constructs. Due to the combined antifungal, osteoproliferative, and biodegradable properties, PCL-based 3D scaffolds enriched with silver showed good potential for bone tissue engineering and offer a promising strategy as an ideal anti-adhesive and anti-biofilm tool for the reduction in prosthetic joints of infections caused by Candida spp. by using antimicrobial molecule-targeted delivery.

Keywords: Candida albicans; Candida auris; Saos-2 cells’ osteogenic differentiation; anti-adhesive/antifungal properties; biofilm inhibition; calcium phosphates; poly(ε-caprolactone)-based biomaterial; silver.

Figure 1

FESEM micrographs of PCL-based scaffolds: pure PCL construct, 50× (A), 1% silver added to a PCL-based construct, 50× (B), BCP/PCL construct at 50× (C), and 100× (D) magnification.

Figure 2

FESEM micrograph of BCP/PLC scaffold at lower (A) and higher (B) magnifications. In (B), the fine and homogeneous distribution of calcium phosphate particles inside the PCL polymer matrix is highlighted by the black arrows.

Figure 3

The ICP-OES analysis, revealing the silver’s release into the medium at different incubation time points from PCL-based scaffolds pored with NaCl (A) and NaNO₃ (B) and added with the two different silver concentrations.

Figure 4

(A) Plate view of Alizarin Red S staining of Saos-2, cultured for 12 and 18 days in the presence or absence of ODM, on different PCL scaffolds. (B) Photometric quantification of Alizarin Red S staining of Saos-2 on different PCL scaffolds. * p < 0.05; *** p < 0.001; # p < 0.001. Saos-2 cultured on PCL + Ag scaffolds vs. cells on the respective PCL scaffolds without Ag, in the presence of ODM, are shown. Mean ± SEM of one representative experiment in triplicate. (C) Representative pictures of transmitted light microscopy at 20X magnification of Saos-2, cultured on PCL + Ag scaffolds and stained with Alizarin Red S to identify mineralization. ODM: osteoblast differentiation medium.

Figure 5

Demonstrative images of the inhibition halo assay against C. auris in the presence of the PCL-based samples pored with NaCl: PCL enriched with 1% (A) or 1.2% (B) of silver, and BCP/PCL enriched with 0.79% (C) or 1% (D) of silver.

Figure 6

Demonstrative images of the inhibition halo assay against C. auris in the presence of the PCL-based samples pored with NaNO₃: PCL enriched with 1% (A) or 1.2% (B) of silver, and BCP/PCL enriched with 0.79% (C) or 1% (D) of silver.

Figure 7

Bar charts of the number of adherent C. albicans (A) and C. auris (B) (log₁₀ colony-forming unit, CFU/mL) on the PCL and BCP/PCL- based scaffolds with added lower silver concentrations, produced with either NaCl or NaNO₃ salts, after 24 h of incubation. Results are the mean values ± standard error of the mean (SEM) of at least three independent experiments. ** p < 0.001 vs. PCL or BCP/PCL, via an unpaired t-test.

Figure 8

Bar charts of the number of planktonic C. albicans (A) and C. auris (B) (log₁₀ colony-forming unit, CFU/mL) on the PCL and BCP/PCL-based scaffolds with added lower silver concentrations, produced with either NaCl or NaNO₃ salts, after 24 h of incubation. Results are the mean values ± standard error of the mean (SEM) of at least three independent experiments. * p < 0.05 vs. PCL or BCP/PCL, via an unpaired t-test.

Figure 9

Demonstrative FESEM micrographs representing the presence of C. albicans biofilm on the un-sonicated pure PCL-based scaffolds, obtained by using NaCl salt as a template, at 100× (A) and 1000× (B) magnification.

Figure 10

Demonstrative FESEM micrographs of the rare presence of C. albicans on PCL-based biomaterials, obtained by using NaCl salt as a template enriched with 1% of silver at 1000× magnification (A), and its morphological alteration with filamentous forms (white arrows) at 5000× magnification (B).