Materials

Polycaprolactone powder (PCL; MW 50,000 Daltons; Tm = 58 ̊C) was purchased from Polysciences Inc. PCL with 50,000 Daltons molecular weight was used following the known suitable viscosity of the melting polymer needed for 3DP from previous studies [17, 20]. Timolol maleate (TML) and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM) and foetal calf serum were purchased from Thermo Scientific (Gibco, UK). Penicillin and streptomycin were purchased from Sigma-Aldrich. CellTiter 96® AQueous One Solution Reagent was purchased from Promega (Southampton, UK).

Synthesis of Timolol-loaded Implant by 3D printed process

Tinkercad, an online 3D CAD design tool (Fig. 1a), was used to design scaffold structures, which were then divided to smaller implantable systems. The Biox™ 3D Bioprinter (Cellink, Sweden) with a thermoplastic printhead was employed, using a 22G conical needle (0.41 mm inner diameter) at 1 mm/s speed. PCL only implants were manufactured by adding the powder directly into the thermoplastic printhead without adding any solvent and fill maximum half of the cartridge to ensure the most efficient heating of PCL. Implants containing both PCL and the timolol maleate were added together in thermoplastic cartridge as the powder form and vortexed for 5 min at 60 s intervals. The layer height for printing was set at 0.8 mm and the infill density was set at 0%. Each implant was composed of 2 layers. To achieve good printability, optimisation of the printing pressures and temperatures was required to achieve good flow (data not shown). The PCL extrusion was smooth, and the implant was fully formed at 150 °C and at the pressure of 175 kPa. With the addition of TML, the temperature remained the same; however, the pressure had to be increased to 190 kPa.

Fig. 1

Dimensions of the implant design (A) and the final 3D printed structures: PCL (B), PCL-5%TML (C) and PCL-10%TML (D)

Microscopic evaluation

Light microscopy images of all implants were taken using the Olympus CKX41 inverted microscope with CellSens Standard 1.13 (Build 13,479) software. The surface morphology of the implants was further analysed using a Scanning Electron Microscope (SEM; Hitachi TM3030 SEM, Tokyo, Japan). Images were taken at a magnification of 5000x in the Energy Dispersive X-Ray (EDX) condition, focusing on the successful melting of PCL and the possible presence of TML powder on the implant’s surface.

Thermal properties

The thermal behaviours of the raw material as well as those of the printed implants were studied using the differential scanning calorimeter (DSC) 214 Polyma (NETZSCH-Geratebau GmbH, Wolverhampton, UK). The samples were weighed and then placed in aluminium crucibles. Starting from 25˚C (Room Temperature, RT) the samples were heated up to 300 °C. Scanning rate was 10 K/min, under protection of nitrogen purge gas (flow rate 20.0 mL/min).

The thermal gravimetric analyser (TGA; Q50, TA, USA) was used to explore the influence of varying concentrations of TML on the thermal stability of 3D-printed implants over a progressive increase of temperature. The samples were weighed and placed in aluminium crucibles. The ramp was set at 20 °C/min from RT to 500 °C, a temperature corresponding to the complete degradation of PCL, under the protection of nitrogen purge gas (flow rate 50.0 mL/min) [20].

Fourier-transform infrared spectroscopy (FTIR)

Attenuated Total Reflection FTIR (ATR-FTIR) Nicolet™ iS50 FTIR Spectrometer (Thermo-Fisher Scientific) was used to analyse both powder and 3DP implants to detect any potential chemical interactions or chemical modifications. Each sample spectrum was obtained between 4000 cm and 1 and 400 cm-1. The samples were run at a resolution of 4 cm-1 with 64 scans after background correction.

In vitro drug release test and implants weight changes

TML was analysed by UV spectrophotometer for determination of the in vitro release using a modified established protocol in our labs [21]. Briefly, the 3D printed implant was cut into 1 cm, then the implant section’s weight was measured before being immersed in a vial containing 1 mL of PBS (pH = 7.4) and kept in an incubator at 37 °C [21,22,23,24]. At each time point (e.g., 1 h, 2 h, 4 h, 6 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks), the entire solution was removed and replaced by 1 mL of fresh PBS. Each sample was analysed using a UV-spectrophotometer (Cole-Parmer, Staffordshire, UK) at a wavelength of 297 nm [25]. Samples from all time points were run in triplicates.

The implants were placed in PBS at 37 °C. The initial weight of the implants was measured at day 0. After the drug-releasing experiment, the implants were thoroughly dried, and the weights were determined. The experiment was performed in triplicates.

In vitro biocompatibility study

Trabecular meshwork tissues from glaucoma patients were used to culture human TM cells [26]. The samples were obtained with the patient’s informed consent and all experimental protocols were approved by the West of Scotland Research Ethics Committee (REC 19/WS/0146). The human TM cells were grown in complete culture media consisting of DMEM, 10% foetal calf serum, and 100 U/mL penicillin/ 0.1 mg/mL streptomycin and cultured in incubators at 37 °C with 5% CO2 and 95% humidity. The human TM cells were plated in a 96-well plate at a density of 6.25 × 103 cells per well. The experiment was done in triplicates for each type of implant (PCL, PCL-5%TML, PCL-10%TML) and untreated control cells. The cells were treated with 50 µL complete media and 50 µL media containing the drug solution collected from each implant on day 1, day 3, day 5, day 7, week 2, week 3 and week 4. After 24 h, the cell media was replaced with fresh culture media, followed by the addition of 20 µL of CellTiter 96® AQueous One Solution Reagent. The plate was incubated for 2 h at 37 °C with 5% CO2 and 95% humidity. The plate was then read using a plate reader, PHERAstar FS (BMG LABTECH), set at 490 nm absorbance, and the results were normalised against untreated control cells. The experiment was performed in triplicates.

Statistical analysis

Statistical significance was determined by One-way ANOVA followed by post hoc test. The statistical significance was indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. All data are expressed as mean ± SD.