Ink-jet 3D printing as a strategy for developing bespoke non-eluting biofilm resistant medical devices

Abstract

Chronic infection as a result of bacterial biofilm formation on implanted medical devices is a major global healthcare problem requiring new biocompatible, biofilm-resistant materials. Here we demonstrate how bespoke devices can be manufactured through ink-jet-based 3D printing using bacterial biofilm inhibiting formulations without the need for eluting antibiotics or coatings. Candidate monomers were formulated and their processability and reliability demonstrated. Formulations for in vivo evaluation of the 3D printed structures were selected on the basis of their in vitro bacterial biofilm inhibitory properties and lack of mammalian cell cytotoxicity. In vivo in a mouse implant infection model, Pseudomonas aeruginosa biofilm formation on poly-TCDMDA was reduced by ∼99% when compared with medical grade silicone. Whole mouse bioluminescence imaging and tissue immunohistochemistry revealed the ability of the printed device to modulate host immune responses as well as preventing biofilm formation on the device and infection of the surrounding tissues. Since 3D printing can be used to manufacture devices for both prototyping and clinical use, the versatility of ink-jet based 3D-printing to create personalised functional medical devices is demonstrated by the biofilm resistance of both a finger joint prosthetic and a prostatic stent printed in poly-TCDMDA towards P. aeruginosa and Staphylococcus aureus.

Keywords: 3d printing; Biofilms; Cell instructive behaviour; Ink-jet; Medical devices.

Figure 1. Schematic for developing optimized formulations for ink-jet based 3D-printing.

A-B) Monomer candidates selected for formulation development and optimization. C) A Fujifilm Dimatix DMP-2830 3D printer was used to print samples. The system in this case was equipped with a cartridge ejecting 10 pL drop volumes, utilising up to 16 nozzles. D) On-slide arrays of cuboids were created by ink-jet based 3D-printing for preliminary microbiology biofilm assays using Pseudomonas aeruginosa. E) Cytotoxicity and cell attachment biocompatibility tests on the printed samples were carried out using mouse embryonic fibroblast 3T3 cells to assess biocompatibility of the printed device; F) Attenuation Total Reflectance Infrared Spectroscopy (ATR-IR) was used to quantify the levels of residual acrylate in the specimens made from different ink formulations; G) Mechanical tests were performed by Dynamic Mechanical Analysis (DMA) in tension mode at room temperature; H) Formulations resulting in desirable properties were tested in vivo to ensure that the cell instructive properties were retained in a more complex environment; I-J) The finalized ink formulations were used to print concept devices.

Figure 2. P. aeruginosa biofilm surface coverage and 3T3 mammalian cell based cytotoxicity assay

A) An array of cuboids was printed onto polystyrene slides and bacterial biofilm formation compared with a silicone control; the samples were imaged after incubation with P. aeruginosa (tagged with the red fluorescent protein mCherry) using confocal microscopy. Biofilm formation was assessed over 640 x 640 μm and presented as biofilm coverage (%) over the whole assessment window (mean ± standard deviation, n = 24)(right); statistically significant differences (*p ≤0.001) were determined using a one-way ANOVA with post-hoc Dunnett’s test with respect to the control (right). An example confocal microscopy image of biofilm formation is included on poly-TCDMDA-DMPA-4 (left) and silicone rubber control (right). B) Comparison of 3T3 fibroblast cytotoxicity (%) for the printed cuboid tablets on different days, the test was performed using an LDH assay: mean ± standard deviation with n = 5; statistically significant differences (*p ≤0.05) were sought using a two-way ANOVA with post-hoc Tukey’s test with respect to the control (right); An example of the Live/Dead® cell viability assay on a poly-TCDMDA-DMPA-4 sample illustrating viable cells and proliferation (left).

Figure 3. Assessment of bacterial viability and biofilm formation in vitro and infection in vivo in a mouse foreign body infection model.

A) Bacterial cell viability on printed specimens, RPMI-1640 medium containing the printed sample was inoculated with either P. aeruginosa (left) or S. aureus (right) cells. Intracellular ATP levels were quantified at early (OD600nm = 0.25), mid (OD600nm = 0.5) and late (OD600nm = 0.8) exponential phase using a BacTiter-Glo microbial cell viability assay, NGPDA with 4 wt% of DMPA as initiator was used as a control. Data show mean ± standard deviation, n = 3; B) Bacterial biofilm formation on printed specimens in vitro: the biofilm biomass of P. aeruginosa and S. aureus was measured after 72 h incubation. Error bars equal ± one standard deviation unit, n = 3. Fluorescent micrographs of mCherry-labelled P. aeruginosa (red) and GFP-labelled S. aureus (green) growing on each surface (right). mean ± standard deviation, n = 3. Each image is 610 x 610 μm². C) ink-jet based 3D-printing optimized formulations (TCDMDA-DMPA-4 and TCDMDA-DETX-4) and biomedical grade silicone sections (as controls) were implanted subcutaneously in mice. After inoculation, light emission from bioluminescent P. aeruginosa at the infection site was measured on the day of inoculation. D) Representative bioluminescence outputs overlaid with bright field images of implanted mice infected with P. aeruginosa and captured on days 0 to 4. The implanted devices and surrounding tissues were also removed on day 4 from each animal and the device-associated bioluminescence quantified ex vivo. E) Bioluminescence was normalised to the output on day 0 showing that the printed devices were colonized with considerably lower levels of bacteria compared with the silicone control.

Figure 4. Structural assessment of the infection site and cellular localisation in tissue surrounding the implant: silicone control, TCDMDA-DMPA and TCDMDA-DETX

A) Structural comparison of architectural changes in tissue surrounding the implant (upper). FM1-43 membrane lipid marker (green), DAPI, nuclear/DNA (orange) and wheat germ agglutinin reactive lectin marker (cyan) staining bacterial microcolonies and the infection site; Immunohistochemical localisation (lower) of P. aeruginosa (magenta), CD45 leukocyte lineage cell populations (blue) and CD206 M2 macrophages (yellow), scale bar: 50 μm; B) schematic of the distribution of different cells in the tissue surrounding the implant.

Figure 5. Ink-jet based 3D-printed finger prosthesis and other demonstrators using the developed ink formulations

A) ink-jet based 3D printed finger prosthesis with TCDMDA-DMPA-4, composed of a central hinge region between two stems, scale bars in the SEM images are 2 mm; B) Fluorescence and overlaid fluorescence-brightfield confocal microscopy 3D images showing in vitro biofilm formation imaged using mCherry-labelled P. aeruginosa (red) and GFP-labelled S. aureus (green) on ink-jet based 3Dprinted finger implants with the developed ink formulations. Scale bars represent 200 μm; C) ink-jet based 3D-printed prostatic stent exemplar with TCDMDA-DMPA-4.