Freeze dryer

Cultivated young leaves of C. formosum were collected from a local field in Ubon Ratchathani province, Thailand. The plant was identified by comparison to the voucher specimen BCY No. 022 deposited in the herbarium of the Faculty of Pharmaceutical Sciences, Ubon Ratchathani University. The leaves were dried at 50°C and sifted through a 40-mesh screen sieve. Extraction of the plant powder was performed using a heat-reflux method (38.83% ethanol in water, 59.76°C, 5.50 h) according to the optimized extraction conditions previously reported by the authors.^{3 (link)}
Ethanol was removed under vacuum, and the resulting CFE in aqueous solution was dried in a freeze dryer (Martin Christ GmbH, Osterode, Germany) before use.

Methanol extract of P. elegans stem was supplied by Foreign Plant Extract Bank (no. FBM118-023; Daejeon, Korea). The plant was collected by Sydara K (one of the authors of the paper) in Ham Ao, Laos in 2010 and authenticated by Institute of Traditional Medicine (ITM). A voucher specimen recoded as ‘Korea Research Institute of Bioscience and Biotechnology (KRIBB) 0033580ʹ was deposited in the herbarium of the Korea KRIBB. Briefly, the dried and refined aerial parts of P. elegans (92g) were extracted with 1,000 mL of 99.9% (v/v) methanol using a sonicator (SDN-900H, SD Ultrasonic Cleaner, Seoul, Korea) at 45°C for 3 days (15 min sonication at 1500 W and 40 kHz followed by 2 h standing; repeated 10 times per day). The resultant product was filtered with non-fluorescent cotton, condensed using a rotary evaporator (N-1000SWD, EYELA, Tokyo, Japan) under reduced pressure at 45°C, and lyophilized using a freeze dryer (Christ, Germany). Dimethyl sulfoxide (DMSO) was used as a solvent for lyophilized substances.

Lyophilization of NLC-Eu ensures long term storage of the samples as it enhances the chemical and physical stability of the nanoparticles. Lyophilized NLC-Eu permits the incorporation of these nanoparticles into more stable form, such as capsules and tablets. Lyophilized NLC-Eu was prepared using a freeze dryer (Martin Christ GmbH, Osterode am Harz, Germany). Briefly, 5 mL of NLC-Eu nano-emulsion was poured into a plastic petri dish and frozen at − 80 °C (Thermoforma, Marlotta, USA) for a few hours. The plates were then covered using parafilm, which had several holes poked through it, and subsequently, freeze-dried for 24 h at − 55 °C [26 (link)].

A scheme for the aqueous ethanol washing process is provided in Figure 1. Aqueous ethanol solutions were prepared in advance by mixing water with 96% ethanol in a Schott glass bottle with the correct volume. The ethanol to the total solvent volume ratio is referred to, in this study, as water/ethanol ratios of 0%, 20%, 40%, 60%, 80%, and 100%.
DFSM was mixed with aqueous ethanol solution in a solid/liquid ratio of 1:10 (w/v) and stirred at room temperature (25 °C) for 30 min. Then, the dispersion was centrifuged (20,000× g, 30 min, 25 °C) to separate the supernatant and pellet. The supernatant was collected as the extract, and the pellet was transferred to a fume hood overnight to evaporate the ethanol. Subsequently, the pellet was freeze-dried (Freeze Dryer, Martin Christ, Osterode, Germany) and milled into powder, which is reported in this study as dried soy protein concentrates (SPCs). All the extracts and SPCs processed with varied water/ethanol ratios were prepared in triplicate and stored at 4 °C for further analysis.

To visualize the TEP derived from the precursors (e.g. various alginate blocks), a bright-field microscope (Keyence, Japan) was employed. The fresh sample solutions were prepared prior to observation as described above. In order to visualize TEP with the bright-field microscope, precursor solutions were stained by freshly pre-filtered (0.05 μm polycarbonate filter) alcian blue solution as presented above. Stained samples were then observed under the microscope. For each sample, about 20 images were randomly taken.
The micro-structures of TEP derived from MG-, MM- and GG-blocks at different Na⁺/Ca²⁺ ratios were also observed by a field emission scanning electron microscopy (FESEM) (Jeol JSM-7600F, Japan). Although TEP were freeze dried prior to microscopic observation, this microscopic technique could still provide direct visualizations of evidence of TEP micro-structures. 10–50 mL of sample solutions prepared as described above were filtered through 0.1 μm polycarbonate filters (Whatman, United Kingdom) at a constant pressure of 0.2 bars and was then rinsed by 1 mL of Milli-Q water. Filters with retained alginate blocks were completely freeze-dried completely in a freeze dryer (Christ, Germany) for further examination. All samples were observed at least three times and 8–10 images were randomly recorded each time.

58sBG with a composition of TEOS (6.6 ml), TEP (0.86 ml) and Ca(NO₃)₂ 4H₂O (4.25 g) was prepared by an evaporation–induced self–assembly (EISA) method as previously reported (Tsigkou et al., 2014 (link)). The ink was first prepared before printing. A total of 1.05 g 58sBG, 1.5 g Gel and 0.6 g of SA were added to 10 ml of deionized water at 55°C in a constant temperature system. Then, magnetic and mechanical stirring were used to mix the materials. The obtained ink was transferred to the barrel of a 3D bioprinter (Regenovo, Hangzhou, China). The experimental parameters of the printing process were set as follows: the needle diameter was 0.4 mm, the extrusion pressure was 0.38 MPa, the printing speed was 15 mm/s, the adjacent filaments were 1.2 mm, and the extrusion temperature was 28°C. According to the needs, two different shapes of scaffolds were printed (10 mm × 10 mm × 1.5 mm cubic scaffold for in vitro study, 5 mm diameter and 1.5 mm high cylindrical scaffold for in vivo study). Next, the obtained scaffolds were soaked in 10% CaCl₂ solution and cross–linked for 10 min. Further cross–linking in 0.25% glutaraldehyde solution for 30 min was carried out. After that, scaffolds were washed with distilled water 5 times and soaked in distilled water for 8 h. Finally, scaffolds were frozen at 80°C and dried for 24 h in a freeze dryer (CHRIST, Germany) for further use.