Growth factor-free salt-leached silk scaffolds for differentiating endothelial cells

Abstract

Recently, controllable kinetic assembly was introduced into the salt-leaching process with silk proteins to form scaffolds, which achieved improvement in tuning the micro-structural and mechanical properties. Here, more control of the kinetic assembly of silk in the process was integrated into salt-leaching process, resulting in significant mechanical modification of the scaffolds generated. Both glycerol additions and treatment to concentrate the protein were used to tune hydrophilic interactions during aqueous solution processing and to reduce beta-sheet formation during the salt-leaching process. These new scaffolds showed gradient changes in elastic modulus in the range of 0.9 to 7.9 kPa. Bone marrow mesenchymal stem cells grew well and showed endothelial differentiation behavior on the scaffolds with optimized stiffness. These results indicated that the introduction of silk kinetic assembly provides an additional option for the control of porous silk scaffold properties.

Subtle Regulation of Scaffold Stiffness for the Optimized Control of Cell Behavior

“…The CD curves were obtained in the spectral range from 240 to 190 nm with a 1.0 nm wavelength step. The secondary conformations of the scaffolds were evaluated with Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). − For FTIR, the samples were measured with a Nicolet FTIR 5700 spectrometer (Thermo Scientific, FL, USA) in the wavenumber range 800–2000 cm –1 . The content of various secondary conformations was further analyzed through Fourier self-deconvolution (FSD) of the amide I region with peakfit software.…”

“…Cell differentiation was assessed using immunofluorescence staining and quantification of endothelial gene expression after 28 days. ,,, Scaffolds were sectioned to slices with a depth of about 2 mm and then sterilized via 60 Co γ-irradiation at the dose of 50 kGy before 1 × 10 5 BMSCs cells were cultured on each scaffold. , The BMSCs were cultured in DMEM without growth factors to evaluate the promotion influence of the mechanical cues on cell differentiation into endothelial cells. After 28 days, immunofluorescence staining was performed as follows. ,, The samples were fixed and blocked with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), 1% Triton X-100, and 3% BSA, respectively.…”

“…Cell differentiation was assessed using immunofluorescence staining and quantification of endothelial gene expression after 28 days. ,,, Scaffolds were sectioned to slices with a depth of about 2 mm and then sterilized via 60 Co γ-irradiation at the dose of 50 kGy before 1 × 10 5 BMSCs cells were cultured on each scaffold. , The BMSCs were cultured in DMEM without growth factors to evaluate the promotion influence of the mechanical cues on cell differentiation into endothelial cells. After 28 days, immunofluorescence staining was performed as follows. ,, The samples were fixed and blocked with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), 1% Triton X-100, and 3% BSA, respectively. The blocked samples were incubated with the anti-CD31 primary antibody overnight (Abcam, Cambridge, MA, USA), conjugated with secondary fluorescence antibody pAB to Ms IgG (FITC, Abcam, Cambridge, MA, USA) for 2 h, followed by staining with rhodamine–phalloidin (Sigma-Aldrich, St. Louis, MO, USA) and 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) to visualize the cytoskeletons and nucleus.…”

Lotus-Root-Like Microchanneled Collagen Scaffold

“…In particular, the leaching strategy has garnered attention in the fabrication of porous scaffolds. Xiao et al integrated a salt leaching process for scaffolds comprising silk proteins . The resulting scaffold exhibited high degrees of porosity, which preceded increased cellular penetration.…”

Biofabrication of Gelatin Tissue Scaffolds with Uniform Pore Size via Microbubble Assembly

“…However, uneven distribution of pore size results in inhomogeneity in porosity and mechanical properties all the way along the scaffold . Phase separation, gas foaming, salt leaching, and freeze drying techniques are conventional porous scaffold fabrication methods in which all of the porosity and scaffold architecture cannot be controlled precisely. Besides, rapid prototyping techniques such as 3D printing, selective laser sintering, stereolithography, or fused deposition modeling, which emerged in the last decade have the capability to control accurate pore positioning; however, each of the techniques is still costly compared to conventional techniques.…”