An immobilized liquid interface prevents device associated bacterial infection in vivo

Abstract

Virtually all biomaterials are susceptible to biofilm formation and, as a consequence, device-associated infection. The concept of an immobilized liquid surface, termed slippery liquid-infused porous surfaces (SLIPS), represents a new framework for creating a stable, dynamic, omniphobic surface that displays ultralow adhesion and limits bacterial biofilm formation. A widely used biomaterial in clinical care, expanded polytetrafluoroethylene (ePTFE), infused with various perfluorocarbon liquids generated SLIPS surfaces that exhibited a 99% reduction in S. aureus adhesion with preservation of macrophage viability, phagocytosis, and bactericidal function. Notably, SLIPS modification of ePTFE prevents device infection after S. aureus challenge in vivo, while eliciting a significantly attenuated innate immune response. SLIPS-modified implants also decrease macrophage inflammatory cytokine expression in vitro, which likely contributed to the presence of a thinner fibrous capsule in the absence of bacterial challenge. SLIPS is an easily implementable technology that provides a promising approach to substantially reduce the risk of device infection and associated patient morbidity, as well as health care costs.

Keywords: Implant; Infection; In vivo; Perfluorocarbon liquids; Polytetrafluoroethylene; SLIPS.

Figure 1. In vitro characterization of ePTFE-SLIPS and anti-bacterial adhesion behavior

(A) Slippery function of SLIPS was measured by tilting angle assay. (B) Reflection confocal microscopy images of lubricant-infused surfaces submerged over varying intervals in PBS or air. (C) SEM images of ePTFE or ePTFE-SLIPS after two days in culture with S. aureus (Scale bar: 10 μm). Red arrow: bacteria. (D) Colony-forming units (CFU) of ePTFE or ePTFE-SLIPS after exposure to S. aureus for two days. (E) CFU of ePTFE or ePTFE-SLIPS incubated in 50% rat serum for varying intervals and subsequently exposed to S. aureus for 48h. Error bars represent mean ± s.d. from at least 3 replicates, *p < 0.05, ePTFE vs PFPE, PFPH and PFD.

Figure 2. Macrophage behavior on ePTFE-SLIPS substrates

(A) Attachment of macrophages was measured by crystal violet (CV) staining after a 1 h incubation. (B) Macrophage viability was measured after a 24 h culture period by Alamar blue reduction. (C) Macrophage phagocytosis was visualized by confocal microscopy. CellTrace Violet-labeled macrophages (blue fluorescence) were exposed to Syto-9-labeled S. aureus (green fluorescence) for 30 min. MØ: macrophages; SA: S. aureus. Red arrow highlights engulfed bacteria. (D) Flow cytometry of bacterial phagocytosis by macrophages after a 1 h incubation period at 37°C or 4°C. (E) Phagocytosis was calculated by subtracting macrophage-associated fluorescence intensity at 4°C from that measured at 37°C. (F) The effect of SLIPS on bactericidal potential of macrophages. Macrophages were incubated with S. aureus for 2 h and viability was examined by a colony forming unit assay. Error bars represent mean ± s.d. from at least 3 replicates (*p < 0.05).

Figure. 3. Bacterial resistance of ePTFE-SLIPS in vivo

(A) Schematic of in vivo model. Circular implants (d = 6 mm) were surgically placed in the subcutaneous tissue on the dorsal side of rat. S. aureus (2.6 × 10⁷ CFU, 50 μL) was injected 24 h later into the implant site. Three days after bacterial challenge, both the implant and local tissue were harvested. (B) Infection rate three days after inoculation. Implants were removed, minced, and sonicated in Luria-Bertani broth and cultured for 20 h at 37°C. Implants were considered infected if broth was turbid (as measured by OD600) and S. aureus was present on an agar plate of the culture. Infection rate (%) was calculated by dividing the number of infected implants by the total number of implants (*p < 0.05). (C) SEM images of retrieved implants three days after bacterial inoculation (Blue arrow: leukocytes; Yellow arrow: matrix; Red arrow: bacteria highlighted in green; Pink arrow: red blood cell; Scale bar: 10 μm). (D) Gram stain of implant-tissue interface (S. aureus dark purple, Red arrow: bacteria, *implant pocket, Scale bar: 100 μm).

Figure. 4. Bacterial burden in peri-implant tissue

(A) Gram stain of peri-implant tissue (S. aureus dark purple, Red arrow: bacteria, Scale bar: 100 μm). (B) Bacterial CFU in local tissue three days and 7 days (C) after inoculation. Local tissue was collected, digested, diluted and plated on agar plate to count CFU, which was normalized by tissue weight. Results are presented from individual implants. (D) Infection rate was calculated at day 7 by dividing the number of infected tissue samples by total sample number.

Figure 5. Histological staining of implants and peri-implant tissue

Three days after inoculation, whole tissue blocks were harvested for hematoxylin and eosin staining. (A,D) ePTFE alone. (B,E) S.aureus (SA) alone. (C,F) ePTFE+SA. (G,J) ePTFE+PFPE+SA. (H,K) ePTFE+PFPH+SA. (I,L) ePTFE+PFD+SA. (A-C, G-I) Magnification 4x. Scale bar: 1 mm. (D-F, J-L) Magnification 20x. Scale bar: 100 μm (*implant pocket). Red arrows highlight immune cell infiltration.

Figure 6. Flow cytometric analysis of inflammatory response within the vicinity of the implant and the peri-implant tissue

Three days after bacterial challenge, implants and surrounding tissue were harvested and digested with collagenase. Flow cytometry was performed with anti-CD45 (A, D), anti-neutrophil (B, E) and anti-macrophage antibodies (C, F). Results are presented from individual implants combined from at least two independent experiments (*p < 0.05).

Figure 7. Host response to SLIPS at day 7

ePTFE or ePTFE-SLIPS implants (d = 6 mm) were harvested at 7 days. (A) Masson's trichrome staining demonstrated a substantially thinner capsule surrounding the ePTFE-SLIPS implant (Yellow line indicates capsule, *implant pocket, Scale bar: 500 μm). (B) Quantification of capsule thickness. (C, D) Effect of SLIPS on macrophage cytokine expression. Macrophages were cultured on tissue culture plastic (TCP), ePTFE or SLIPS-treated ePTFE for 18 hours and IL-1β and IL-6 measured by q-PCR. Error bars represent mean ± s.d. from at least 3 replicates, *p < 0.05.