Nanofibrous and planar fibrinogen scaffolds
were prepared by our previously described salt-induced self-assembly
approach.43 (link),44 (link) Briefly, 100% clottable fibrinogen from
human plasma (Merck, Darmstadt, Germany), dissolved in 10 mM NH4HCO3 (Carl Roth GmbH, Karlsruhe, Germany) in deionized
H2O, was dialyzed overnight against the same solution using
a cellulose membrane dialysis tube (cutoff 14 kDa; Sigma-Aldrich,
Darmstadt, Germany) to obtain a fibrinogen solution. Fifteen millimeter
glass coverslips (VWR, Darmstadt, Germany) were cleaned with piranha
solution (3:1 of 95% sulfuric acid (H2SO4)/30%
hydrogen peroxide (H2O2)) before treatment with
5% (3-aminopropyl)triethoxysilane (APTES, Sigma) in ethanol (C2H5OH) overnight. Unbound APTES was removed by washing
in pure ethanol three times for 5 min, before storing the coverslips
dry for further use.
To obtain fibrinogen nanofibers, 5 mg mL–1 of the fibrinogen solution was dried in the presence
of 2.5× phosphate-buffered saline (PBS, ThermoFisher, pH 7.4)
on APTES-modified coverslips. Planar fibrinogen scaffolds were obtained
by drying 5 mg mL–1 of fibrinogen in 5 mM NH4HCO3. Drying was performed in a custom-built climate
chamber at a relative humidity of 30% and temperature of 24 °C for 12 h.
To maintain the stability
of scaffolds in an aqueous environment
for further cell culture studies, the scaffolds were cross-linked
in formaldehyde (FA) vapor for 2 h after placing them in a sealed
beaker. The FA vapor was generated by placing 1 μL of 37% FA
solution (Applichem GmbH, Darmstadt, Germany) per cubic centimeter
and letting it evaporate in the sealed beaker. After cross-linking
and devaporizing for another 30 min, all samples were washed 3 × 15 min with deionized water. We have extensively
characterized the nanofibrous topography with dense, porous nanofiber
networks of these fibrinogen nanofiber scaffolds as well as the smooth
topography of the planar fibrinogen scaffolds previously.43 (link),45 (link)To obtain un-cross-linked adsorbed fibrinogen substrates,
the so-called
physisorbed fibrinogen scaffolds, a slightly modified protocol from
a previously published method was used.46 (link) Fifteen millimeter glass coverslips (VWR) were cleaned with piranha
solution (3:1 of 95% sulfuric acid (H2SO4)/30%
hydrogen peroxide (H2O2)). A 0.1 mg mL–1 fibrinogen solution in PBS (Thermo Fisher, pH 7.4) was then dried
on the piranha-cleaned coverslips at 4 °C overnight. Subsequently,
the scaffolds were washed three times for 5 min with PBS. The physisorbed
fibrinogen scaffolds displayed a flat and smooth topography (seeSupporting Information , Figure S4), also described
earlier.46 (link)Nanofibrous, planar, as
well as physisorbed fibrinogen scaffolds
were placed in wells of nontreated Corning Costar 24-well plates (Sigma)
and sterilized for 30 min using the UV light of a laminar flow cabinet
(ESI Flufrance) to further use them in cell culture experiments.
were prepared by our previously described salt-induced self-assembly
approach.43 (link),44 (link) Briefly, 100% clottable fibrinogen from
human plasma (Merck, Darmstadt, Germany), dissolved in 10 mM NH4HCO3 (Carl Roth GmbH, Karlsruhe, Germany) in deionized
H2O, was dialyzed overnight against the same solution using
a cellulose membrane dialysis tube (cutoff 14 kDa; Sigma-Aldrich,
Darmstadt, Germany) to obtain a fibrinogen solution. Fifteen millimeter
glass coverslips (VWR, Darmstadt, Germany) were cleaned with piranha
solution (3:1 of 95% sulfuric acid (H2SO4)/30%
hydrogen peroxide (H2O2)) before treatment with
5% (3-aminopropyl)triethoxysilane (APTES, Sigma) in ethanol (C2H5OH) overnight. Unbound APTES was removed by washing
in pure ethanol three times for 5 min, before storing the coverslips
dry for further use.
To obtain fibrinogen nanofibers, 5 mg mL–1 of the fibrinogen solution was dried in the presence
of 2.5× phosphate-buffered saline (PBS, ThermoFisher, pH 7.4)
on APTES-modified coverslips. Planar fibrinogen scaffolds were obtained
by drying 5 mg mL–1 of fibrinogen in 5 mM NH4HCO3. Drying was performed in a custom-built climate
chamber at a relative humidity of 30% and temperature of 24 °C for 12 h.
To maintain the stability
of scaffolds in an aqueous environment
for further cell culture studies, the scaffolds were cross-linked
in formaldehyde (FA) vapor for 2 h after placing them in a sealed
beaker. The FA vapor was generated by placing 1 μL of 37% FA
solution (Applichem GmbH, Darmstadt, Germany) per cubic centimeter
and letting it evaporate in the sealed beaker. After cross-linking
and devaporizing for another 30 min, all samples were washed 3 × 15 min with deionized water. We have extensively
characterized the nanofibrous topography with dense, porous nanofiber
networks of these fibrinogen nanofiber scaffolds as well as the smooth
topography of the planar fibrinogen scaffolds previously.43 (link),45 (link)To obtain un-cross-linked adsorbed fibrinogen substrates,
the so-called
physisorbed fibrinogen scaffolds, a slightly modified protocol from
a previously published method was used.46 (link) Fifteen millimeter glass coverslips (VWR) were cleaned with piranha
solution (3:1 of 95% sulfuric acid (H2SO4)/30%
hydrogen peroxide (H2O2)). A 0.1 mg mL–1 fibrinogen solution in PBS (Thermo Fisher, pH 7.4) was then dried
on the piranha-cleaned coverslips at 4 °C overnight. Subsequently,
the scaffolds were washed three times for 5 min with PBS. The physisorbed
fibrinogen scaffolds displayed a flat and smooth topography (see
earlier.46 (link)Nanofibrous, planar, as
well as physisorbed fibrinogen scaffolds
were placed in wells of nontreated Corning Costar 24-well plates (Sigma)
and sterilized for 30 min using the UV light of a laminar flow cabinet
(ESI Flufrance) to further use them in cell culture experiments.
