*2.6. Total Cefazolin Loading Content*

Specimens (10 mm × 10 mm) were immersed in a bottle containing 5 mL of artificial cerebrospinal fluid (aCSF; 6.279 g/L sodium chloride, 0.216 g/L potassium chloride, 0.353 g/L calcium chloride, 0.488 g/L magnesium chloride, 1.932 g/L sodium bicarbonate, 0.358 g/L disodium hydrogen phosphate and 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) 11.915 g, pH 7.4 [27]) and incubated for 24 h at 37 ◦C in an incubator. The specimen was taken out and aCSF was collected and filtered. The absorbance of the collected aCSF was then measured by using a UV–Vis spectrophotometer (Perkin Elmer) at a wavelength of 270 nm and compared with a linear equation of the calibration curve (cefazolin in aCSF, R-squared = 0.9996) to quantify the amount of released cefazolin from the sample. The standard cefazolin solutions ranged from 0.02, 0.05, 0.10, 0.25, 0.5, 1, 5, 10, 15, 20, 25 to 30 μg/mL. The collected sample was further dried at 40 ◦C and dissolved in 5 mL dimethylformamide (DMF) in a shaking incubator for 24 h at 37 ◦C, 100 rpm. The DMF solution was then filtered through a silica glass filter and the amount of cefazolin was quantified by using a UV–Vis spectrophotometer in a similar manner as that for aCSF but using the linear calibration curve of cefazolin in DMF solutions (R-squared = 0.9867) instead. The total amount of cefazolin in the samples was calculated by combining the measured cefazolin amount in the aCSF and that in DMF solution. The measurement was performed in triplicate and the result was expressed as the ratio of the total amount of cefazolin (mg) to the mass (g) of each specimen.

#### *2.7. In Vitro Cefazolin Release Study*

Specimens (10 mm × 10 mm) were prepared and each sample was placed in a plastic bottle containing 5 mL of aCSF and secured in place with a frame sheet to ensure that both sides of the sample were totally immersed in the solution. This was then incubated in an incubator at 37 ◦C. Every 24 h, the specimen was removed, slightly blotted with tissue paper and placed in a new bottle containing 5 mL of fresh aCSF. The remaining solution was collected and filtered with a 0.45 μm syringe filter (Corning). The absorbance of cefazolin in the collected solution was then measured by using a UV–Vis spectrophotometer (Perkin Elmer) at a wavelength of 270 nm and compared with a linear equation of the prepared calibration curve (cefazolin in aCSF; R-squared = 0.9996) as previously described to quantify the amount of cefazolin released from the sample.
