Quattro esem

Scaffolds were fixed in 10% formalin acetate overnight at 4°C, washed 2x with water, soaked overnight in OCT compound, frozen, and sectioned at 5 µm. Cell distribution throughout the scaffold was visualized using a DAPI nuclear stain (D1306, Invitrogen).
Pore architecture and protein adsorption to the pore surface were visualized with scanning electron microscopy.^{9 (link),18 (link)} Scaffolds were fixed in freshly prepared Karnovsky’s fixative solution overnight at 4°C, washed 2x with water, bisected, and dehydrated in increasing concentrations of ethanol. Following dehydration, samples were critical point dried (Supercritical AutoSamdri-931, Tousimis Research Corp, Rockville, MD), fixed to stubs with silver paste, sputter coated with gold (Pelco SC-7 Auto Sputter Coater), and imaged using a scanning electron microscope (Quattro ESEM, Thermo Fisher, Newington, NH).

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (Nicolet 6700 FTIR spectrometer, Thermo Electron Co., MA, USA) was employed to analyze the structural changes of the membrane during the preparation processes. The absorbance of the spectrum was acquired in the range of 400–4000 cm⁻¹ with a resolution of 2 cm⁻¹. Scanning electron microscope (SEM) images were captured by a field emission-SEM (FE-SEM) of Quattro ESEM (Thermo Fisher Scientific, MA, USA). The quantification of –SH groups on the membrane was achieved by using Ellman’s reagent [35 (link)]. Specifically, precisely weighted 2 mg of LDNM was immersed in 2.5 mL of PBS buffer, then 50 μL of 10 mg/mL Ellman’s reagent was added into the solution. The mixture was incubated at room temperature for 10 min, then the color intensity of the solution was scanned in a UV-vis spectrophotometer (Evolution 600, Thermo Fischer). The absorbance at 412 nm (A₄₁₂) was recorded and converted to the –SH concentration in a unit of mM in solution (C_SH) according to a self-established calibration curve (A₄₁₂ = 1.6959C_SH + 0.0576, R²= 0.9996). ¹H Nuclear Magnetic Resonance (NMR) was performed on a Brucker 400 MHz NMR (Bruker Co., MA, USA) by using DMSO-d₆ as a solvent.