Gentamicin sulphate

Brassica nigra seeds (Benkor, Velký Borek, Czech Republic), Capsella bursa-pastoris leaves (Hanus, Nitra, Slovakia) and Lavandula angustifolia leaves (Juvamed, Rimavská Sobota, Slovakia) were purchased as commercial products. Origanum vulgare leaves were collected in summer from the meadow at the University of Veterinary Medicine and Pharmacy campus in Košice, Slovakia. Berberis vulgaris root was obtained in spring from a private garden in Byster, Slovakia. Collected leaves and roots were dried up to constant weight in the dark at room temperature. Silver nitrate (AgNO₃, 99.9%, Mikrochem, Pezinok, Slovakia), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma Aldrich, St. Louis, MO, USA), Folin–Ciocalteau reagent (Sigma Aldrich, St. Louis, MO, USA), methanol (p.a. 99.5%, Mikrochem, Slovakia), sodium carbonate (>99%, Mikrochem, Pezinok, Slovakia), gallic acid (97.5–102.5%, Sigma Aldrich, St. Louis, MO, USA) and gentamicin sulphate (Sigma-Aldrich, St. Louis, MO, USA) were used without further purification.

Starting 3 days before adoptive T cell transfer, mice were placed on a high dose gavage treatment of 200 μL antibiotic, consisting of 1 mg/mL ampicillin (anhydrous, 96.0%–102.0%, Sigma-Aldrich), gentamicin sulphate (Sigma-Aldrich), metronidazole (Metronidazole BioXtra, Sigma-Aldrich), neomycin (trisulfate salt hydrate, Sigma-Aldrich), and 0.5 mg/mL vancomycin (vancomycin hydrochloride, Streptomycesorientalis, potency ≥900 μg/mg, Sigma-Aldrich) diluted in PBS. On day 0, mice received 2 × 10⁶ WT CD4⁺CD3⁺CD25^– T cells i.v. and were monitored for change in weight over 15 days.

The antibacterial properties of the samples were evaluated by the agar well diffusion method. The tested bacteria (Staphylococcus aureus CCM 4223 and Escherichia coli CCM 3988) were obtained from the Czech collection of microorganisms (CCM). A few hours before the antibacterial activity tests, the powders containing AgNPs were mixed with the distilled water (20 mg of the powder was put into 1 mL water) and sonicated. Just before the inoculation on the agar plate, the suspensions were mixed again using a laboratory stirrer to ensure the most homogeneous distribution of the solid particles in water. Subsequently, 50 µL of samples prepared in the form of a suspension were introduced into the wells and gentamicin sulphate (Sigma-Aldrich, Saint-Louis, Missouri, USA), with a concentration of 30 mM, was used as a positive control. The antibacterial activity is expressed as the relative inhibition zone diameter (RIZD), where the activity of the positive control is considered as 100% and the activity of the tested substances compared with it. Other details of antibacterial activity evaluation in this study are the same as reported previously [26 (link)].

The decellularisation procedure for Study 2 was as described for Study 1 but with the acetone treatment included as a fat reduction step. The following changes were then subsequently introduced. A 3 h bioburden reduction step was included immediately after the acetone treatment using either peracetic acid (0.1%; v/v [Sigma]) or an antibiotic solution (PBS containing 0.05 mg ml⁻¹ vancomycin hydrochloride, 0.5 mg ml⁻¹ gentamicin sulphate, 0.2 mg ml⁻¹ polymyxin [all from Sigma]), both at room temperature. In addition, each bioburden reduction step was investigated with and without the terminal PAA treatment described in Study 1. This was to determine any interacting effects it may have with the bioburden steps on the mechanical properties of the tissue.
Hence, seven groups were investigated in Study 2:•

Native (untreated)

•

DC2+TPAA

•

DC2−TPAA

•

DC2+PAAbio+TPAA

•

DC2+PAAbio−TPAA

•

DC2+ABbio+TPAA

•

DC2+ABbio−TPAA.

DC2: decellularisation process with acetone permanently included (i.e. DC2=DC1+ACE), TPAA: terminal peracetic acid treatment, PAAbio: peracetic acid bioburden treatment, ABbio: antibiotic bioburden treatment. + and − denote ‘with’ and ‘without’ respectively.
The steps investigated and the processes involved are shown in Fig. 1(b).

Triton X-100, tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTS), sodium alginate, gentamicin sulphate, sodium acetate trihydrate, phosphate buffer solution (PBS) tablets, o-phthaldialdehyde reagent solution (OPA), piperazine, and 1,6 hexanediol diacrylate were purchased from Sigma-Aldrich, UK. Cyclohexane, n-hexanol, ammonium hydroxide (35%), diethyl-ether, di-chloro methane (DCM), ethanol, methanol, glacial acetic acid, and iso-propanol were purchased from Fisher Scientific, UK. All reagents were stored according to manufacturer’s guidelines and used as received. The bone cements brands used were Cemex (Tecres S.p.A., Italy) and Palacos R (Heraeus Medical GmbH, Germany).
Acetic acid-sodium acetate buffer (0.1 M, pH 5) was prepared mixing sodium acetate trihydrate (CH₃COONa·3H₂O) (0.1 M) and acetic acid (CH₃COOH) (0.1 M) solutions 3:7 and stirred, with pH checked and adjusted in the range 5.0 ± 0.1. PBS solution (pH 7.4) was prepared according to manufacturer’s guidelines.

Gentamicin Sulphate (Sigma) [44 (link)], in a sterile solution of 0.9% saline, pH to 7 (adjusted with 1 M NaOH), was administered by subcutaneous injection once daily to male rats at doses of 0, 10, 25 and 50 mg/kg/day for 7 days (maximum of 1 mL per site and no more than 4 sites in any 24 hour period).