## 2 Materials and Methods
### Characterizations of Crosslinking  
The degree of crosslinking is expressed as the percentage of crosslinking present in a sample with respect to the dry, non-crosslinked control sample cut from the same sheet of electrospun material [25]. For the sake of investigating the degree of crosslinking, 2,4,6-trinitrobenzenesulfonic acid solution (TNBS) was used [29; 33]. The rationale for the test is a kind of colorimetric assay. As the degree of crosslinking increases, the number of free aminos in the collagen would decrease followed by a parallel decrease in the number of binding sites which are available for TNBS to interact with. Three samples with an average mass 1.8 mg were punched from each crosslinking treatment and untreated samples. Then, the samples were placed into a solution of 1.0 mL 4% (\(w/v\)) sodium bicarbonate and 1.0 mL freshly prepared 0.5% (\(v/v\)) TNBS in deionized water and heated at 40 \({}^{\circ}\)C for 2 h. Subsequently, 3 mL of 6 M HCl was added to each sample and incubated for an additional 1.5 h at 60 \({}^{\circ}\)C. Finally, the solution was diluted with 5.0 mL of deionized water. Aliquots of equal volume from each sample were read by a Perkin Elmer Lambda 25 UV-visible spectrophotometer operating at 345 nm. The degree of crosslinking was calculated as follows:  
\[Cross-linked(\%)=\left(1-\frac{Abs_{c}}{mass_{c}}/\frac{Abs_{ac}}{mass_{ac}} \right)\times 100 \tag{2}\]  
where \(c\) is the crosslinked sample, \(nc\) is the non-crosslinked sample. \(Abs\) is absorbance at 345 nm. The unit of mass is mg.  
A scanning electronic microscope (SEM, TM3000, Hitachi Ltd., Tokyo, Japan) and weight loss ratio were used to investigate the water stability of samples under physiological conditions. The samples (2 \(\times\) 2 cm\({}^{2}\)) were immersed in phosphate buffered saline (pH = 7.4) at 37 \({}^{\circ}\)C for up to 7 days with the solution refreshed every day. Then, the samples were freeze dried to constant weight. In order to characterize the stability performance of morphology under physiological conditions, small squares (0.5 cm \(\times\) 0.5 cm) were cut from the samples and gold coated by sputtering. The samples were observed by SEM at an accelerated voltage of 15 kV and a working distance of 10 mm. The weight loss ratio was calculated by weighing the samples before and after incubation in PBS. \[Weight-loss(\%)=\frac{W_{i}-W_{d}}{W_{i}}\times 100 \tag{3}\]  
where \(W_{i}\) is the initial dry weight of sample before incubation and \(W_{d}\) is the dry weight of sample after incubation in PBS for a certain time period. Five samples were used per time point. In order to reveal the effect of crosslinking and detect the mechanical properties in an environment, which is more closely to the end use, the tensile properties in dry and hydrated states were examined. Tensile properties of the electrospun fibrous membranes were determined with an electronic Strength Tester (XF-1A, Xin Xian Instrument Co., Ltd., Shanghai, China) using a low force load cell of 100 cN capacity at ambient temperature of 25 \({}^{\circ}\)C and humidity of 60%. Strip-shaped specimens (5 \(\times\) 50 mm\({}^{2}\)) were tested three times at a loading speed of 20 mm/min and then the thickness was measured with a digital thickness meter having a sensitivity of 1 \(\upmu\)m. For the hydrated state, the crosslinked samples were immersed in PBS for 24 h at room temperature prior to testing without changing the testing conditions. Finally, the elastic modulus, ultimate tensile stress, and ultimate tensile strain were calculated from the resultant stress-strain curves. Fourier transform infrared spectroscopy (FTIR) was applied to analyze the surface functional group changes. Spectra of collagen/PEO nanofibers before and after crosslinking were collected by a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). All spectra were recorded in the wavelength range 4000-600 cm\({}^{-1}\).