2 propanol

Titanium isopropoxide (TTIP; reagent grade, 99%, Aldrich, (Palo Alto, CA, USA), 2-propanol, nitric acid (HNO₃), and distilled water were used for TiO₂ nanoparticles synthesis. Graphene oxide was synthesized using graphite (325 mesh, Alfa-Aesar, Tewksbury, MA, USA), sulfuric acid (H₂SO₄), potassium permanganate (KMnO₄), hydrogen peroxide (H₂O₂), and 2-propanol (Merck, Burlington, MA, USA). CS beads were prepared using chitosan (from shrimp shells) with a molecular weight of Mv 144.000, measured by capillary viscometry (using an Ubbelohde 0C viscometer, Schott, Barcelona, Spain). K and a constants for CS in the solvent used (acetic acid 0.3 M + sodium acetate 0.2 M) at 25 °C are 0.074 mL/g and 0.76, respectively [81 (link)], for the calculation of the molecular weight with the Mark-Houwink-Sakurada equation (Equation (1)): $\left[\eta \right]=K{\left(Mv\right)}^a$
The degree of deacetylation determined by ¹H-NMR using a 400 MHz AVANCE II spectrometer (Bruker, Billerica, MA, US) at a temperature of 300 K and elemental analysis of the CS was 89%. The sample was dissolved in D₂O with two drops of trifluoroacetic and 3-(trimethylsilyl)propionic acid-d₄ as reference salt. The elemental analysis was performed using a Flash EA 1112 system (Thermo Electron, Waltham, MA, US).

For measurement of lipid accumulation, 3T3L1 adipocytes were washed with PBS for 1 min, treated with 4% paraformaldehyde in PBS (Nacalai Tesque) at 37 °C for 30 min, washed with 60% (vol/vol) 2-propanol (Nacalai Tesque), and then dried at 37 °C for 15 min. The fixed cells were stained with 0.3% (wt/vol) oil red O (Sigma-Aldrich) in 60% (vol/vol) 2-propanol at 37 °C for 30 min, washed with distilled water three times, and then incubated with 100% 2-propanol at room temperature for 1 min. Absorbance at 490 nm was measured by using an iMark microplate reader (Bio-Rad, Hercules, CA, USA).

CMC was synthesized according to ref.[27 (link)]. First, 13.5 g NaOH was dissolved in 100 ml of solvent comprising 20 ml water and 80 ml 2-propanol (Sigma-Aldrich Inc., St. Louis, MO, USA), to which 10 g chitosan (85% deacetylated, Sangon Co., Ltd. (Shanghai, China) was gradually added under stirring (1000 rpm). This mixture was allowed to swell and alkalize at 25°C for 2 h. Next, 15 g monochloroacetic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) dissolved in 20 ml 2-propanol was added dropwise to the mixture in 30 min. The activated chitosan was reacted with the acid for 4 h at 50°C, which was stopped by addition of 200 ml of 70% ethanol. After that, the resulting precipitate was desalted by dialysis and then dried under reduced pressure at 25°C to obtain CMC powder.
CMC gel was first prepared by mixing 2.5 g CMC into 40ml water under stirring (1000 rpm) until the CMC powder was completely dissolved. Then, 0.498 g K₂HPO₄ was added into the CMC gel under stirring (500 rpm). Next, 0.555 g CaCl₂ was added into 10 ml deionized water and this solution was added dropwise into the CMC gel under stirring for 5 min to form CMC/ACP gel. The final concentrations of calcium and phosphate ions were 100 mM and 60 mM, respectively. This gel was immediately frozen at -80°C for 2 h and then lyophilized in vacuum freeze dryer (Lgj-B10, Beijing, China) for 6 h to form CMC/ACP scaffolds.