Spatial patterning of nanofibrous collagen scaffolds modulates fibroblast morphology

Abstract

Current knowledge about cell-biomaterial interactions is often based on two-dimensional (2D) cell culture systems like protein-coated glass slides. However, such smooth surfaces cannot mimic the nanofibrous environment of the native extracellular matrix (ECM). It is therefore a major challenge to transfer the results from 2D surfaces to 3D protein scaffolds with biomimetic nanofiber architecture. To understand the influence of different protein topographies on the cell response we introduce a new process to fabricate binary collagen scaffolds of variable thickness with spatially controlled regions of nanofibrous and smooth topography. We used pH-induced self-assembly to prepare collagen nanofibers with diameters between 130 and 150 nm on glass surfaces, which were partly covered with a polymer mask. After cross-linking with glutaraldehyde, smooth collagen films were prepared on the remaining glass regions. Atomic force microscopy revealed a much lower surface roughness of smooth collagen compared to nanofibers. Subsequently, we studied the viability, morphology and migration of 3T3 fibroblasts on both collagen topographies. We found small, elongated fibroblasts with few, long filopodia on collagen nanofibers whereas large, flat fibroblasts with many short filopodia were observed on smooth collagen. Actin stress fibers on collagen nanofibers were substantially reduced in comparison to smooth collagen. Live cell tracking revealed that fibroblasts on thin nanofibrous collagen migrated faster than on smooth collagen. In summary, binary collagen scaffolds enabled us for the first time to study cell responses to topographical cues on a single protein scaffold. In future, it will be intriguing to transfer our patterning process to other proteins to study fundamental principles of topography-dependent cell recognition processes.

Effect of Collagen Nanofibers and Silanization on the Interaction of HaCaT Keratinocytes and 3T3 Fibroblasts with Alumina Nanopores

“…Therefore, 2.5 mg/mL of collagen was found to yield a very dense fiber network compared to 1 mg/mL of collagen, where the fiber density was reduced (see Figure C,D). This trend is also in good agreement with our previous study of nanofibrous collagen scaffolds on glass slides . Diameters of individual fibers ranged approximately from 108 ± 17 nm to 162 ± 31 nm for 2.5 and 1 mg/mL collagen, respectively.…”

“…Independent of the collagen concentration, 3T3 proliferation rates on collagen-modified AAO substrates were found to range around 40–50% of the glass control during all time points. Previously, 3T3 fibroblasts were found to be viable on both nanofibrous and smooth collagen scaffolds maintained up to 72 h in culture . Also, in other reports, 3T3 fibroblasts were observed to show increased migration on nanotopographies due to a reduction of the accessible surface area .…”

“…Since no significant change in the fiber formation/morphology was observed for these smaller pores (see Supporting Information Figure S2), the 200 nm membranes were chosen for further studies due to the larger pore sizes, which would help in better gas/nutrient exchange during cell culture experiments. Overall, our SEM analysis shows that the assembly of collagen could be successfully transferred from solid substrates such as glass or polydimethylsiloxane (PDMS) to the highly porous carrier substrates of anodic alumina membranes. The finding that bulk and porous substrate materials are equally suited to enable protein fiber assembly will be an important prerequisite for providing biochemical stimuli on nanoporous scaffold materials and to establish multifunctional porous biomaterials for wound healing and other biomedical applications. , …”

“…Collagen nanofibers were assembled on AAO membranes as previously described . In brief, a stock solution of 5 mg/mL of collagen type I from calf skin (Sigma, Steinheim, Germany) in 0.5% acetic acid was prepared.…”

“…Moreover, we studied the morphology of fixated cells on the different substrate types with SEM. Cell samples were fixated after proliferation analysis using 2% GA solution, followed by an ethanol exchange series as described earlier . Prior to SEM analysis, both sample types were sputter-coated with approximately 7 nm of gold using a Bal-Tec SCD 005 sputter system (Leica Microsystems).…”