Antibacterial Albumin-Tannic Acid Coatings for Scaffold-Guided Breast Reconstruction

Abstract

Infection is the major cause of morbidity after breast implant surgery. Biodegradable medical-grade polycaprolactone (mPCL) scaffolds designed and rooted in evidence-based research offer a promising alternative to overcome the limitations of routinely used silicone implants for breast reconstruction. Nevertheless, as with any implant, biodegradable scaffolds are susceptible to bacterial infection too, especially as bacteria can rapidly colonize the biomaterial surface and form biofilms. Biofilm-related infections are notoriously challenging to treat and can lead to chronic infection and persisting inflammation of surrounding tissue. To date, no clinical solution that allows to efficiently prevent bacterial infection while promoting correct implant integration, has been developed. In this study, we demonstrated for the first time, to our knowledge that the physical immobilization of 1 and 5% human serum albumin (HSA) onto the surface of 3D printed macro- and microporous mPCL scaffolds, resulted in a reduction of Staphylococcus aureus colonization by 71.7 ± 13.6% and 54.3 ± 12.8%, respectively. Notably, when treatment of scaffolds with HSA was followed by tannic acid (TA) crosslinking/stabilization, uniform and stable coatings with improved antibacterial activity were obtained. The HSA/TA-coated scaffolds were shown to be stable when incubated at physiological conditions in cell culture media for 7 days. Moreover, they were capable of inhibiting the growth of S. aureus and Pseudomonas aeruginosa, two most commonly found bacteria in breast implant infections. Most importantly, 1%HSA/10%TA- and 5%HSA/1%TA-coated scaffolds were able to reduce S. aureus colonization on the mPCL surface, by 99.8 ± 0.1% and 98.8 ± 0.6%, respectively, in comparison to the non-coated control specimens. This system offers a new biomaterial strategy to effectively translate the prevention of biofilm-related infections on implant surfaces without relying on the use of prophylactic antibiotic treatment.

Keywords: 3D printing; albumin; antibacterial coating; bacterial infection; polycaprolactone; scaffold; tannic acid.

FIGURE 1

Morphological characterization of macro- and microporous 3D printed scaffolds (A) Scanning electron microscopy images showing the surface of extruded mPCL/Sugar scaffolds (i–iii) after printing, red arrows indicate sugar crystals and (iv–vi) after leaching out the sugar particles for 15 days in order to generate micro-sized pores, which are indicated with red arrows. (B) μCT evaluation of microporous mPCL scaffolds showing the 3D representation of the (i) segmented scaffold struts after sugar leaching as well as the (ii) segmented micropores. (iii) 2D distribution of micropores across a virtual plane through the scaffold, struts are shown in clear blue and micropores in dark blue, red arrows indicate local interconnectivity of pores. (iv) Equivalent diameter distribution for the micropores present on the surface of and within the scaffolds.

FIGURE 2

Scanning electron microscopy images showing (A) the surface of HSA/TA coated scaffolds after fabrication, from lower (i) to higher (iii) magnifications, as well as (iv) resin-embedded cross sections evidencing distinctive coating thicknesses. (B) SEM images of uncoated and coated scaffolds after incubation in PBS, DMEM and DMEM + 10% FBS at 37°C, for 3 days. Red arrows indicate adhered agglomerates on the coated surfaces. Scale bars: 20 μm.

FIGURE 3

Surface characterization of untreated and treated surfaces. (A) Wide and (B) high resolution O 1s, C 1s, and N 1s, XPS scan spectra (C) FTIR spectra evidencing the presence of hydroxyl and amide groups on the surface of treated scaffolds, demonstrating successful immobilization of HSA and TA on the surface.

FIGURE 4

In vitro evaluation of treated scaffolds in a 2D zone of inhibition assay against (A) gram-positive S. aureus and (B) gram-negative P. aeruginosa. 1%HSA/10%TA- and 5%HSA/1%TA- treated scaffolds exhibited antimicrobial activity against both bacteria strains seen in the form of a clear zone around the samples in the agar plate. (C) Comparison of inhibition zones by antibiotic-loaded disks (+Control) and HSA/TA-coated scaffolds. Data shown as mean ± SD, n = 8. (^∗∗∗p < 0.001).

FIGURE 5

3D in vitro evaluation of antibacterial effectiveness of coated scaffolds against S. aureus in suspension. (A,B) Uncoated scaffolds showed extensive bacterial colonization by S. aureus, while (C,E) scaffolds coated with 1% and 5%HSA showed significantly fewer adherent bacteria on the surfaces. Scaffolds coated with 1% and 5%HSA and stabilized with 10% and 1%TA, respectively, (D,F) did not only evidenced reduced bacteria colonization, but also showed morphological changes on the bacterial membrane suggesting possible membrane disruption. (G) Number of viable colony forming units of S. aureus recovered from the scaffolds surface. Data shown as mean ± SD, n = 6. (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001); scale bars: 2 μm.