We have presented a new approach for the fabrication of 3D micro-structured covalently assembled protein gradients. An enabling aspect of the MPE approach is that the protein concentration can be changed without altering the topography, affording the ability to examine the roles of biochemical and spatial cues (separately and together) that contribute to the measured response. We further suggest this approach overcomes some limitations of previous synthetic methods to create insoluble gradients. For example, Plummer et al.32
fabricated FN gradients through creating a gradient of deposited gold unto which FN was covalently bound. They observed preferential fibroblast attachment to higher concentrations, however, the pattern was 2 dimensional and the FN was not linked together. Photocrosslinking of the FN as achieved through the MPE technique may be more biomimetic.
Our approach also provides increased mechanical stability. This is evidenced by the observation that after 3 days in culture, based on phase contrast and fluorescence imaging, the gradients showed no clear degradation (not shown). Additionally, the fabricated structures are mechanically robust with respect to either water flow or dehydration/rehydration. This stability arises because the fabricated protein structures are covalently linked to each other and to the BSA monolayer, which is then strongly adsorbed to the organosilane monolayer, which is covalently bound to the glass substrate. In contrast, in micro-contact printing, protein molecules adhere to each other and to the substrate via much weaker Van de Waals forces.
The microscope-based approach further permits the synthesis of structures with sub-micron topographic features, presenting cells with analogous topographic cues that are present in the native ECM. Moreover, since full length purified proteins are used cell integrins are exposed to the appropriate protein binding sites, e.g., the RGD/PHSRN sequences on FN. For example, in prior work, through the “stick and grip” method,17
we observed that the β1
integrin was active in dermal fibroblast binding to MPE fabricated FN structures (unpublished results). Additionally, we found high specificity of the monoclonal HFN7.1 antibody to MPE crosslinked human FN.29
While our long-term motivation lies in tissue engineering applications, here we focused our attention on studying the fundamental cell–matrix interactions. Specifically the FN gradients were used as substrates to compare the fibroblast response to changing protein concentrations in the presence of micro-structured topography. This is of interest as the role of concentration gradients on cell response is not completely understood. For example, using immobilized gradients created through microfluidics, Kenis and co-workers18,19
showed that the primary factor in governing cell migration was the local concentration, rather than the concentration slope. In contrast, other reports have cited the importance of local slope.5
Our data for 3T3 fibroblasts on FN suggests the importance of local concentration, as the cell orientation and elongation at the constant concentrations (representative images and analysis in and respectively), were similar to those observed on the gradient ().
We note that the net fibroblast response further depends on the combination of topographic features and protein concentration. While some alignment exists for cells on the BSA gradient, which provided only topographic cues, this is a weak effect compared to FN, where the average orientation angles were 38 and 6° respectively. In strong contrast, the alignment and elongation data for the cells shown in were statistically different although the topographies were similar (continuous linear structures of the same height and width) but with different FN concentrations, revealing the contribution of the ECM cues. The examples of using a BSA gradient/FN background and FN gradient/FN background further showed how contact guidance and ECM cues can operate together and separately. By contrast, other reports documented that the morphological and cytoskeletal properties of cells on submicron patterned structures resulted primarily from contact guidance. For example, Nealey and co-workers37,38
reported that both epithelial cells and keratocytes showed a high degree of alignment on silicon grooves and ridges. They also observed highly aligned stress fibers but cells had fewer focal adhesions relative to those adhered distally to the etched patterns.
While we did not find a global statistically significant correlation between the spatial distribution of focal adhesions and FN fibers (with respect to total expression), some focal adhesions were co-localized with the FN structures (examples denoted by arrows in ). It was not expected that the focal adhesions would be solely located on the FN as the BSA monolayer is not a repulsive background and the cells will eventually form new adhesions. This is a different scenario than sometimes reported in the literature where islands or stripes of attractive regions were placed against a cell-repulsive background, and the focal adhesions are expressed solely on the fabricated regions by default.14,16
This observation is consistent with our prior work which showed that the distribution of focal adhesions of fibroblasts adhered on crosslinked BSA microfibers were randomly distributed between these features.29
Our data taken in conjunction with that of Nealey is suggestive that the stress fiber alignment may primarily result from the topographic response, whereas the focal adhesion distribution arises from a combination of topographic and ECM cues.
We need to also consider the role of feature size and spacing on the resulting cell morphology and cytoskeletal features. As the instrument is microscope-based, and MPE is a threshold process, the size of the fabricated features cannot be significantly increased. Specifically, we have not been able to achieve efficient photocrosslinking at lower resolution (which would yield larger fabricated features) than the optics that were used here (0.5 NA), which resulted in lateral diameters of about 700 nm. However, comparisons can be drawn to recent work by Doyle et al.14
Through a combination of printing and ablation, they created 1D “stripes” of varying widths (1–40 μ
m) from FN and examined the spreading of 3T3 fibroblasts. For cells on thinner stripes (e.g., 1 μ
m) they observed a similar uniaxial phenotype to that shown here at high FN concentration. When the widths became larger than the cell size, this elongation was decreased and the morphology approached the typical fibroblast behavior seen on monolayers or in culture. In terms of fiber spacing, we previously showed that when FN and fibrinogen fibers (~700 nm width) were spaced by distances much larger than cell diameters, i.e., 40 μ
m, the effects of alignment and elongation of fibroblasts were decreased relative to those adhered to fibers spaced by 10 μ
m (~one cell diameter).29