Self-assembled nanofiber coatings for controlling cell responses.

Nanofibers are thought to enhance cell adhesion, growth, and function. We demonstrate that the choice of building blocks in self-assembling nanofiber systems can be used to control cell behavior. The use of 2 D-coated, self-assembled nanofibers in controlling lens epithelial cells, fibroblasts, and mesenchymal stem cells was investigated, focusing on gene and protein expression related to the fibrotic response. To this end, three nanofibers with different characteristics (morphology, topography, and wettability) were compared with two standard materials frequently used in culturing cells, TCPS, and a collagen type I coating. Cell metabolic activity, cell morphology, and gene and protein expression were analyzed. The most hydrophilic nanofiber with more compact network consisting of small fibers proved to provide a beneficial 2 D environment for cell proliferation and matrix formation while decreasing the fibrotic/stress behavior in all cell lines when compared with TCPS and the collagen type I coating. This nanofiber demonstrates the potential to be used as a biomimetic coating to study the development of fibrosis through epithelial-to-mesenchymal transition. This study also shows that nanofiber structures do not enhance cell function by definition, because the physico-chemical characteristics of the nanofibers influence cell behavior as well and actually can be used to regulate cell behavior toward suboptimal performance. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2252-2265, 2017.

Effects of peptide ratios in nanofibre-based hydrogels for the prevention of capsular opacification.

PURPOSE: To moderate the capsular opacification (CO) response after lens surgery, an experimental study was performed in which nanofibre-based hydrogels (nanogels) with different ratios of attached peptides were applied to provide extracellular matrix-related cues for lens epithelial cells (LECs) in a porcine eye model. METHODS: The lens content was removed, and the capsules were refilled with nanogel. Lenses were divided into two groups, the first group (n = 34) was refilled with nanogels containing different ratios of two laminin-derived peptides (IKVAV + YIGSR), and the latter group (n = 26) was refilled with nanogel combinations of a fibronectin-derived and a type IV collagen-derived peptide (RGDS + DGEA). Two lenses were refilled with culture medium to investigate the effect of the medium on LECs. After refilling, lenses were extracted and cultured for 3 weeks. Lens epithelial cells (LECs) were assessed for morphology and alpha-smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. RESULTS: Differences were seen in cell morphology between lenses refilled with nanogels with IKVAV + YIGSR and RGDS + DGEA peptides. In nanogels with IKVAV + YIGSR peptides, differences in LEC morphology were largest when ratios between the peptides were unequal, whereas LEC responses from the RGDS + DGEA refilled groups showed variation in LEC morphology dependent on the total quantity of mixed-in peptides. The culture medium did not induce proliferation or transformation of LECs. CONCLUSIONS: Ratios and concentrations of cell adhesion-mediating peptides both can direct the LEC response, depending on the adhesion molecules of origin, by influencing LEC proliferation and transformation. Nanogels with incorporated peptides may be tuned towards CO prevention.

Nanofiber-based hydrogels with extracellular matrix-based synthetic peptides for the prevention of capsular opacification.

Nanofiber-based hydrogels (nanogels) with different, covalently bound peptides were used as an extracellular environment for lens epithelial cells (LECs) in order to modulate the capsular opacification (CO) response after lens surgery in a porcine eye model. Lenses were divided into 15 groups (n = 4 per group), the lens content was removed and the empty capsules were refilled with nanogel without peptides and nanogels with 13 combinations of 5 different peptides: two laminin-derived, two fibronectin-derived, and one collagen IV-derived peptide representing cell adhesion motifs. A control group of 4 lenses was refilled with hyaluronan. After refilling, lenses were extracted from the porcine eye and cultured for three weeks. LECs were assessed for morphology and alpha smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. Compared to hyaluronan controls, lenses filled with nanogel had less CO formation, indicated by a lower αSMA expression (P = 0.004). Microscopy showed differences in morphological cell response within the nanogel refilled groups. αSMA expression in these groups was highest in lenses refilled with nanogel without peptides (9.54 ± 11.29%). Overall, LEC transformation is reduced by the presence of nanogels and the response is improved even further by incorporation of extracellular matrix peptides representing adhesion motifs. Thus, nanomaterials targeting biological pathways, in our case interactions with integrin signaling, are a promising avenue toward reduction of CO. Further research is needed to optimize nanogel-peptide combinations that fully prevent CO.

2D and 3D self-assembling nanofiber hydrogels for cardiomyocyte culture.

Collagen is a widely used biomaterial in cardiac tissue engineering studies. However, as a natural material, it suffers from variability between batches that can complicate the standardization of culture conditions. In contrast, synthetic materials are modifiable, have well-defined structures and more homogeneous batches can be produced. In this study, several collagen-like synthetic self-assembling nanofiber hydrogels were examined for their suitability for cardiomyocyte culture in 2D and 3D. Six different nanofiber coatings were used in the 2D format with neonatal rat cardiomyocytes (NRCs) and human embryonic stem-cell-derived cardiomyocytes (hESC-CMs). The viability, growth, and functionality of the 2D-cultured cardiomyocytes were evaluated. The best-performing nanofiber coatings were selected for 3D experiments. Hydrophilic pH-sensitive nanofiber hydrogel coassembled with hyaluronic acid performed best with both NRCs and hESC-CMs. Hydrophilic non-pH-sensitive nanofiber hydrogels supported the growth of NRCs; however, their ability to promote attachment and growth of hESC-CMs was limited. NRCs also grew on hydrophobic nanofiber hydrogels; however, the cell-supporting capacity of these hydrogels was inferior to that of the hydrophilic hydrogel materials. This is the first study demonstrating that hydrophilic self-assembling nanofiber hydrogels support the culture of both NRCs and hESC-CMs, which suggests that these biomaterials hold promise for cardiac tissue engineering.

Taming membranes: functional immobilization of biological membranes in hydrogels.

Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, protein translocation activity by the membrane embedded protein conducting channel of bacteria, SecYEG, in association with the soluble motor protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

Nanowires formed by the co-assembly of a negatively charged low-molecular weight gelator and a zwitterionic polythiophene.

Conjugated organic nanowires have been prepared by co-assembling a carboxylate containing low-molecular weight gelator (LMWG) and an amino acid substituted polythiophene derivative (PTT). Upon introducing the zwitterionic polyelectrolyte PTT to a basic molecular solution of the organogelator, the negative charges on the LMWG are compensated by the positive charges of the PTT. As a result, nanowires form through co-assembly. These nanowires are visualized by both transmission electron microscopy (TEM) and atomic force microscopy (AFM). Depending on the concentration and ratio of the components these nanowires can be micrometers long. These measurements further suggest that the aggregates adopt a helical conformation. The morphology of these nanowires are studied with fluorescent confocal laser scanning microscopy (CLSM). The interactions between LMWG and PTT are characterized by steady-state and time-resolved fluorescence spectroscopy studies. The steady-state spectra indicate that the backbone of the PTT adopts a more planar and more aggregated conformation when interacting with LMWG. The time- resolved fluorescence decay studies confirm this interpretation.

Beta-cyclodextrin host-guest complexes probed under thermodynamic equilibrium: thermodynamics and AFM force spectroscopy.

The rupture forces of individual host-guest complexes between beta-cyclodextrin (beta-CD) heptathioether monolayers on Au(111) and several surface-confined guests were measured in aqueous medium by single molecule force spectroscopy using an atomic force microscope. Anilyl, toluidyl, tert-butylphenyl, and adamantylthiols (0.2-1%) were immobilized in mixed monolayers with 2-mercaptoethanol on gold-coated AFM tips. For all guests and for all surface coverages, the force-displacement curves measured between the functionalized tips and monolayers of beta-CD exhibited single, as well as multiple, pull-off events. The histograms of the pull-off forces showed several maxima at equidistant forces, with force quanta characteristic for each guest of 39 +/- 15, 45 +/- 15, 89 +/- 15, and 102 +/- 15 pN, respectively. These force quanta were independent of the loading rate, indicating that, because of the fast complexation/decomplexation kinetics, the rupture forces were probed under thermodynamic equilibrium. The force values followed the same trend as the free binding energy Delta G degrees measured for model guest compounds in solution or on beta-CD monolayers, as determined by microcalorimetry and surface plasmon resonance measurements, respectively. A descriptive model was developed to correlate quantitatively the pull-off force values with the Delta G degrees of the complexes, based on the evaluation of the energy potential landscape of tip-surface interaction.