Page 76 - IJB-9-3

International Journal of Bioprinting 3DP hydrogels to combat antibiotic-resistant bacteria

2.9. 3D printing of GelMA-PLGA NPs hydrogels continue challenging the bacteria with each antibiotic,
The inks were transferred to a 30-mL sterile syringe and 2 µL of the well with the highest antibiotic concentration
kept in the cartridge (3D Bioplotter, Envisiontec GmbH, which had allowed visible growth (i.e., ½ MIC) was added
Gladbeck, Germany) for 1.5–2 h to stabilize the temperature to a new plate with serial dilutions of the antibiotics as
of the ink. The cartridge and the printing bed temperature described above. This was repeated for 25 passages.
were set at 26°C and 10°C, respectively. Computer-aided The antimicrobial resistant strains were tested for
design (CAD) models were designed using Fusion360 antibiotic susceptibility by VITEK analysis (VITEK,
(Autodesk, Inc., San Rafael, CA, United States) and sliced BioMerieux, Marcy l’Etoile, France) to confirm the
using Perfactory RP (Envisiontec GmbH). Squares of 20 × resistance to each antibiotic.
20 mm were designed, with a distance between fibers of
0.5 mm. The angle between layers was set at 0/90 degrees, 2.12. Assessment of antibiotic resistance stability
and a stainless-steel dispensing tip G23 12.7 mm (Nordson, To assess the stability of the antimicrobial resistance,
Duluth, GA, United States) length was used. the bacteria were cultured on blood agar plates without
antibiotics for 6 passages. Then, an MIC assay combined
Four different GelMA-NPs hydrogels were 3D printed
in sterile conditions, i.e., (i) GelMA-C-NPs, (ii) GelMA-Rif- with a minimum bactericidal concentration (MBC; the
concentration that kills ≥99.9% of the bacteria after 24 h)
NPs, (iii) GelMA-Van-NPs, and (iv) GelMA-Rif-Van-NPs, assay was performed for each bacterial strain for each
a combination of GelMA-Rif-NPs and GelMA-Van-NPs specific antibiotic at each passage. To assess the MBC,
inks in alternated layers (Figure 1d, Table 3). Four-layer duplicate 10 µL aliquots from the 10-fold serial diluted
hydrogels were printed for all groups. The 3D-printed antibiotic suspensions were cultured on blood agar plates
hydrogels were UV-crosslinked with 25 mW/cm at 37°C.
2
UV light 365 nm (BlueWave 75 UV Light Curing Spot
Lamp, Torrington, CT, United States) for 90 s. 2.13. Kirby–Bauer agar diffusion assay
A Kirby–Bauer agar diffusion assay was performed to
2.10. Bacterial strains determine the zone of inhibition (ZOI) for the different S.
[39]
S. aureus RN4220 , S. aureus RN4220 Rif-resistant (S. aureus strains with the GelMA hydrogels containing either
R
aureus RN4220 Rif ), S. aureus RN4220 Van-resistant (S. C-NPs, Rif-NPs, Van-NPs, or hydrogels of GelMA with
aureus RN4220 Van ), and S. aureus AMC 201 [40,41] were Rif-NPs and GelMA with Van-NPs.
R
used for this study. S. aureus RN4220 Rif and S. aureus
R
RN4220 Van were selected by exposing S. aureus RN4220 First, a bacterial suspension of each strain was prepared
R
to increasing concentrations of Rif and Van, respectively, by suspending 5 colonies in 5 mL of PBS. Then, Mueller
for 25 passages (see section 2.11). Prior to experiments, Hinton agar plates (Oxoid) were inoculated with a swab
bacteria from frozen stocks were grown overnight at 37°C soaked in the bacterial suspension. The hydrogels were
on sheep blood agar plates (BioMerieux, Marcy l’Etoile, placed on the agar plate and incubated overnight at
France). For each experiment, fresh subcultures were made 37°C. The next day, the ZOIs (in mm) were measured
in tryptic soy broth (TSB; Oxoid, Hampshire, UK) at 37°C. at 4 positions, at the middle of each side of the square
hydrogel. To assess the antimicrobial activity over time, the
2.11. Development of antibiotic resistance in procedure was repeated daily for 10 days, by transferring
S. aureus RN4220 the hydrogels after each incubation to freshly inoculated
S. aureus RN4220 was cultured overnight at 37°C at agar plates.
200 rpm in TSB. Then, 100 µL of the overnight culture was
transferred to 5 mL TSB and this suspension was incubated 2.14. In vitro bacterial adhesion assay
for 3 h at 37°C at 120 rpm to reach the exponential growth An in vitro bacterial adhesion assay was performed to
phase. In wells of a 96-well flat-bottom plate (Greiner bio- assess the antimicrobial activity of the hydrogels and to
one, Monroe, NC, United States), serial dilutions from quantify the number of adhered bacteria. The S. aureus
128 µg/mL to 0.125 µg/mL of the antibiotics (Rif or Van) strains were cultured to the mid-logarithmic growth phase
in 90 µL were added to 10 µL of bacterial inoculum (final in TSB at 37°C and 120 rpm, and subsequently diluted in
6
concentration of 10 colony forming units (CFU) /mL) and TSB to 1 × 10 CFU/mL. Of each bacterial strain, 1 mL of
6
incubated overnight at 37°C and 120 rpm. All incubations the suspension was added to separate GelMA hydrogels
were done in duplicates. (n= 6 per type of hydrogel) in 24-well plates and incubated
overnight at 37°C and 120 rpm. The suspensions were
The minimum inhibitory concentration (MIC), i.e., collected in separate tubes for bacterial quantification, and
the lowest concentration of antibiotic that prevents visible the hydrogels were gently washed twice with demineralized
bacterial growth, was determined for each antibiotic. To water, subsequently placed in 1.5-mL Eppendorf tubes with

Volume 9 Issue 3 (2023) 68 https://doi.org/10.18063/ijb.683