Electrospun nanofibrous SF/P(LLA-CL) membrane: a potential substratum for endothelial keratoplasty

Figures & data

Table 1 Primers used for quantitative polymerase chain reaction

Genes	Accession number	Forward (5′–3′)	Reverse (5′–3′)	Annealing temperature (°C)	Product size (base pairs)
AQP1	NM_198098	CTGGGCATCGAGATCATCGG	ATCCCACAGCCAGTGTAGTCA	60	158
Caspase-3	NM_004346	CATGGAAGCGAATCAATGGACT	CTGTACCAGACCGAGATGTCA	60	139
CLCN3	NM_001243372	GAGTTTTGCCTTTCTTGCAGTT	GAAAAGATATTTCCGCAGCAAC	60	234
Col-4	NM_000092	GCCGGTTGAATTTCCTTCATAT	ACCTTTAACGGCACCTAAAATGA	60	232
Col-8	NM_001850	GGAGATGCCCCACTTGCA	GCCGGTTGAATTTCCTTCATAT	60	72
CTNND1	NM_001085465	AGGATCCAGCAAACGATACAGT	AGGTCAGCTATGGCAGAAAGAG	60	244
GAPDH	NM_002046	GAGATCCCTCCAAAATCAAGTG	GAGTCCTTCCACGATACCAAAG	60	284
IL-1	NM_000576	TGTACGATCACTGAACTGCA	GAAGTCAGTTATATCCTGGC	60	433
IL-8	NM_000584	TCTTGGCAGCCTTCCTGATT	TCCAGACAGAGCTCTCTTCCATC	60	211
Na^+K^+ATPase	NM_001160233	ACGGCAGTGATCTAAAGGACAT	CAGAAGAATCATGTCAGCAGCTTG	60	257
SLC4A4	NM_001098484	GCTTGCAGATTACTACCCCATC	TTGAACACTCCTTCTTCGACAA	60	209
VDAC3	NM_005662	ATAAGTTGGCTGAAGGGTTGAA	TTCTGTGACAGTTTGGATTTGG	60	235
ZO-1	NM_003257	TTCTGAGGCCTGTAACCATTT	AATTGGATACCACTGGGCATAG	60	245

Abbreviations: AQP1, aquaporin 1; caspase-3, apoptosis-related cysteine peptidase; CLCN3, voltage-sensitive 3 chloride channel; Col-4, collagen type IV; Col-8, collagen type VIII; CTNND1, cadherin-associated protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1, interleukin-1; IL-8, interleukin-8; Na⁺K⁺ATPase, Na⁺/K⁺ transporting, alpha 1 polypeptide; SLC4A4, sodium bicarbonate cotransporter member 4; VDAC3, voltage-dependent anion channel 3; ZO-1, zonula occludens-1.

Table 2 Basic features of the blended nanofibrous membrane

SF:P(LLA_CA) (8% w/v)	Nanofiber diameter (nm)	Specimen thickness (μm)	Contact angle at 20 seconds (degrees)	Tensile strength (MPa)
100:0	147±24	30±9.53	38.36±0.41	1.90±0.75
75:25	226±31	56±5.12	49.03±0.67	2.39±0.22
50:50	255±37	62.5±7.93	62.93±0.50	5.29±0.66
25:75	226±24	56±4.20	71.58±0.15	9.39±0.69
0:100	542±107	128±6.65	125.78±0.02	7.47±0.38

Note: Data are shown as the mean ± standard deviation, unless otherwise specified.

Abbreviations: SF, silk fibroin; P(LLA-CL), poly(l-lactic acid-co-ε-caprolactone).

Figure 1 Transmission of light in nanofibrous membranes with different blend ratios.

Notes: General transmission of light is shown in (A). Scanning electron microscopy results are shown in (B–F). (B) 100% SF, (C) 75% SF, (D) 50% SF, (E) 25% SF, and (F) 0% SF. Scale bar, 5 μm. Accurate transmission percentage compared with the control group is shown in (G).

Abbreviation: P(LLA-CL), poly(l-lactic acid-co-ε-caprolactone); SF, silk fibroin.

Figure 2 Cells adhering to different blend ratio nanoscaffolds.

Notes: Violet crystals reflect the HCEC-B4G12 numbers and are recorded in (A). After dissolving the violet crystals in dimethyl sulfoxide, absorbance at 490 nm was measured, and the results are shown in (B). **P<0.01.

Abbreviations: SF, silk fibroin; P(LLA-CL), poly(l-lactic acid-co-ε-caprolactone); OD, optical density; HCEC, human corneal endothelial cells.

Figure 3 Cell viability and proliferation on different SF/P(LLA-CL) membranes.

Notes: The first row of (A) shows the results of the live/dead kit test, and few dead cells can be seen. The second row of (A) shows Ki-67 protein staining, and the third row of (A) shows BrdU staining under laser scanning confocal microscopy. The scale bar indicates 50 μm in (A). The histogram of the positive percentage of Ki-67 protein staining is shown in (B). The histogram of the positive percentage of BrdU staining is shown in (C). *P<0.05, **P<0.01.

Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; SF, silk fibroin; P(LLA-CL), poly(l-lactic acid-co-ε-caprolactone).

Figure 4 Monolayer formation on different SF/P(LLA-CL) membranes. HCEC-B4G12 cells were cultured on different membranes for 1 week for monolayer formation.

Notes: Scanning electron microscopic images showed that the B4G12 cells could form a monolayer on all membranes and on glass coverslips (control group), as shown in (A). ZO-1 staining of monolayers is shown in (B). Scale bar, 20 μm.

Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; SF, silk fibroin; P(LLA-CL), poly(l-lactic acid-co-ε-caprolactone); HCEC, human corneal endothelial cells; ZO-1, zona occludens-1.

Figure 5 Gene expression on different membranes.

Notes: (A) Expression of seven functional correlation genes was tested. (B) Two extracellular matrices, collagen IV and collagen VIII, were detected. (C) Interleukin-1 (an inflammatory cytokine) and caspase-3 (an apoptosis-related gene) were tested.

Abbreviations: AQP1, aquaporin 1; CLCN3, voltage-sensitive 3 chloride channel; ZO-1, zona occludens-1; Col-4, collagen type IV; Col-8, collagen type VIII; CTNND1, cadherin-associated protein; IL-1, interleukin-1; Na⁺K⁺ATPase, Na⁺/K⁺ transporting, alpha 1 polypeptide; SLC4A4, sodium bicarbonate cotransporter member 4; VDAC3, voltage-dependent anion channel 3; ECM, extracellular matrix.