Heraeus cytoperm 2

To evaluate the capacity of biomaterials to release calcium ions in an aqueous environment, the liquid extracts from specimens were prepared according to ISO 10993-5:2009 standard recommendations [37 ]. The samples of curdlan-based biomaterials and KALTOSTAT^® specimens were placed into a 12-well plate and EMEM medium with an addition of 2% FBS was added (extraction ratio was equal to 0.1 g of biomaterial/1 mL of EMEM medium). After 24 h incubation at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air (Heraeus cytoperm 2, Thermo Scientific, Waltham, MA, USA), the liquid extracts were collected. The EMEM medium incubated without biomaterials served as a control extract. The concentration of calcium ions in collected solutions was evaluated using the Calcium ion detection kit in accordance with the manufacturer’s protocol.

The cell culture experiments were performed using normal human skin fibroblasts, i.e., BJ cell line (ATCC, London, UK), as it is known as a good model for the evaluation of wound healing in vitro [33 (link),38 (link),39 (link),40 (link),41 ]. The cells were grown in EMEM medium with an addition of 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. According to ATCC directions, BJ cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air (Heraeus cytoperm 2, Thermo Scientific, Waltham, MA, USA). To assess fibroblast response to the tested biomaterials, the liquid extracts from the samples of curdlan-based biomaterials and KALTOSTAT^® specimens were prepared according to ISO 10993-5:2009 standard directions [37 ], as described in Section 2.8. For evaluation of cell viability, extracts were prepared using EMEM supplemented with 2% FBS, while extracts obtained in EMEM with an addition of 10% FBS was applied for estimation of cell proliferation. As a control extract, appropriate EMEM medium (with 2% or 10% FBS) incubated without biomaterials was utilized. The ISO 10993-5:2009 standard guidelines [37 ] are commonly applied for the evaluation of biological properties in vitro of biomaterials with biomedical potential [33 (link),34 (link),42 (link),43 (link),44 (link)].

Ability of biomaterials to transmit water vapor was evaluated according to procedure described by Rezvanian et al. [28 (link)] with some modifications. Prior to test, 5 g of silica gel with indicator was put into glass vials (diameter of mouth of vial was 1 cm) and dried for 24 h at 60 °C (Drying oven SUP-65, Wamed). At the same time, three independent biomaterial samples (n = 3) were immersed in SWF (24 h, room temperature) in order to obtain totally soaked samples. Then, the vials (with dry silica gel) were weighted. The biomaterials were taken out of solution, blotted with tissue paper, and precisely mounted on the mouth of vials using rubber rings to prevent any water vapor influx through the boundary. The vials with biomaterials were transferred to the incubator (Heraeus cytoperm 2, Thermo Scientific, Waltham, MA, USA) for 24 h (37 °C, 95% relative humidity). After this time, the biomaterials from vials were removed and the vials with wet silica gel were weighted. The water vapor transmission rate (WVTR) was calculated using the following formula: $\mathrm{WVTR} \left(\mathrm{g}/{\mathrm{m}}^2/\mathrm{day}\right)=\frac{\mathrm{Ww}-\mathrm{Wd}}{\mathrm{S}}$
where Ww is a weight of vial with wet silica gel (after 24-h incubation), Wd is a weight of vial with dry silica gel (before incubation), and S is surface area of mouth of vial (m²).