2.3 Results
2.3.1 Physico-chemical properties of silk films
2.2.9.4 Collagen gel contraction assay
The functional contractility attained by porcine SMCs with respect to biophysical cues presented by different films were assessed using collagen gel contraction assay, following previously published protocols [246]. Briefly, porcine SMCs were seeded on different films at a seeding density of 6250 cells/cm2 and cultured for 4 days in SMC growth media supplemented with 10%
(v/v) FBS. Thereafter, the cells were detached using trypsin and resultant cell suspension was seeded on to preformed collagen gels (collagen-I, rat tail, Gibco, USA) following the manufacturer’s protocol in 96 wells plate at density 105 cells/mL. After 24h, the extent of collagen gel contraction was assessed using Image-J by measuring the area of gel before contraction and after contraction.
2.2.10 Statistical analysis
All of the statistical analyses were performed using OriginPro 8 (Origin lab Corporation, USA) following one-way analysis of variance (ANOVA) and Tukey’s test. All the experiments were performed at-least for n=3, unless otherwise specified. Significant levels were analyzed at 95%
and 99% confidence and represented as #p<0.05 and ##p<0.01. All data is represented as mean ± standard deviation.
were visualized under FESEM (Figure 2.2B). AA films showed rougher surface whereas smoother surface and edges were observed for BM films.
Another crucial determining factor is surface contact angle or surface hydrophobic/hydrophilic nature that governs cellular adhesion, spreading and functionality. We further investigated the water contact angle for both of silk protein types. Both silk films were hydrophilic owing to the fact that their water contact angles were well below 90°; however, their degree of hydrophilicity was significantly different. BMF films showed approximately 1.43 times higher contact angle as compared to AAF films, making it moderately hydrophilic than AAF type (Figure 2.2C). We further analyzed the contact angles for patterned silk films along and perpendicular to the direction of microgrooves. Again, a similar trend was observed and AAP films showed relatively higher degree of hydrophilicity with less contact angle. The contact angle values for various silk films are summarized in Table 2.2.
Table 2.2. Contact angles of silk films.
Sample Contact angle (in degrees)
BMF 66.92 ± 1.75
AAF 46.76 ± 1.39
BMP PAR 68.41 ± 1.28
BMP PER 64.98 ± 1.56
AAP PAR 40.93 ± 1.02
AAP PER 39.58 ± 1.87
Figure 2.2. Surface characterization of silk films. (A) Atomic force microscopic images of flat and patterned silk films showing surface roughness. Surface plot profile of patterned films is also illustrated indicating the groove depth and peak-to-peak distance. (B) FESEM micrographs of silk films demonstrating surface topography and patterning. (C) Water contact angles of flat silk films (BMF and AAF) and patterned silk films (BMP and AAP in both parallel (PAR) and perpendicular (PER) to the microgroove direction). Images are showing droplet micrographs in contact with silk films. Contact angle values mentioned in the table are representative of at least three different spots onto silk film surfaces.
Furthermore, mechanical properties of silk films were analyzed. Only water vapor annealed flat silk films were subjected for this analysis. Stress-strain curves suggested higher slope of AAF
films as compared with BMF (Figure 2.3A). The representative load-displacement curves are also shown in Figure 2.4. AAF films were stronger/stiffer and showed ~2-fold higher modulus (32.54
± 9.51 MPa) than BMF films (16.79 ± 0.51 MPa) (Figure 2.3B, p<0.01). A similar trend was observed for stress at failure (3.09 ± 0.65 MPa vs 8.54 ± 1.04 MPa for BMF vs AAF films respectively) (Figure 2.3C, p<0.01); however, no significant difference was observed for strain at failure (0.25 ± 0.08 MPa vs 0.30 ± 0.12 MPa for BMF vs AAF films respectively) (Figure 2.3D).
Tensile cyclic testing of silk films was also performed to obtain hysteresis behavior. BMF films did not exhibit much of energy loss between first and last cycle (Figure 2.3E); however, strain hardening and energy gain was observed for AAF films until fifth cycle followed by plateau for last five cycles (Figure 2.3F).
Figure 2.3. Mechanical properties of silk films. (A) Representative stress-strain curves showing the slope of BM and AA flat hydrated films. (B) Modulus of silk films calculated from stress-strain curve slope. (C) Comparison of stress at failure values for both silk films. (D) Comparison of strain at failure values for both silk films. (E) Representative tensile cyclic analysis of BM silk films. (F) Representative tensile cyclic analysis of AA silk films. (##p<0.01, n.s.=not significant)
Figure 2.4. Representative load-displacement curves of hydrated silk films.
Chemical structure analysis was further performed to investigate the secondary structure composition of silk films (Figure 2.5A). Silk fibroin solution (AAS and BMS) and water vapor annealed films (AAF and BMF) were subjected for this analysis. All specimen showed characteristic peaks in amide-I (1610-1660 cm-1), amide-II (1510-1560 cm-1) and amide-III (1210- 1260 cm-1) regions [247]. On comparing the samples before (AAS, BMS) and after (AAF, BMF) water vapor annealing, a blue shift was observed in amide-I region. Alternatively, amide-II and amide-III regions demonstrated a red shift, a characteristic of β-sheet transition after water vapor annealing, making silk films water stable [247]. In order to quantify the secondary structure composition in silk solution and water stable silk films, we de-convoluted the amide-I region between 1600-1700 cm-1 region (Figure 2.5B). The percentage of secondary structures after water vapor annealing is summarized in Table 2.3. The blue shift noticeable in the amide-I spectra of water annealed films meant an increase anti-parallel β-sheet and cumulative β-sheet content was much higher than in fibroin solution indicating water stability of films consistent with previous reports. The higher percentage of β-sheet in non-mulberry silk is attributed to the poly-alanine present in AA.
Table 2.3. Effect of water vapor annealing on silk secondary structure composition.
Secondary structures Percentage (%)
BMS AAS BMF AAF
β-sheets 44.7 58.1 29 33.2
α-helices 7.7 23.1 - 1.7
Loops 36.2 5.9 16.3 -
Antiparallel β-sheets - 1.7 34.9 61.9
Disordered structures 11.3 11.2 19.8 3.2
Figure 2.5. Fourier transform infrared (FTIR) spectra of silk films. (A) Spectrographs showing peak shifts in amide-I, II and III peaks before and after β-sheet induction: BM solution (BMS). AA solution (AAS), films after β-sheet induction (BM film (BMF), AA film (AAF)). (B) Deconvolution of amide-I peaks to determine secondary structure through second order derivative of amide-I spectra for i) BMS, ii) AAS, iii) BMF and iv) AAF.