Introduction: Focal adhesions mediate cellular attachment, migration and mechanotransduction. Molecular level structure organization has been investigated to show how the integrins attach to the cytoskeleton[1] and initiate signaling. Other investigations have shown that substrate patterning is reflected in the localization of focal adhesions mediating cellular attachment to adhesive regions[2]. Electrospun nanofibers present cells with an extracellular matrix like adhesive structures and are known to affect cell differentiation. Despite their ability to promote cell attachment, to modulate the differentiation process, and their common usage as tissue engineering scaffolds, the nature of focal adhesions in cells attaching to nanofibers is not well understood.
Materials and Methods: Electrospun nanofiber scaffolds are fabricated from polymethylmethacrylate (PMMA) dissolved at 25 weight% in nitromethane and collected on a stationary target using a 20cm working distance, 15kV driving voltage, 2.5mL/hr flow rate and an 18G needle. Fibers were diameter reduced using a 55:45 PGMEA:limoline solution. Primary integrin antibodies are from CellSignaling Technologies (4749S). Quantification of immunostaining was conducted using the CellProfiler software (www.cellprofiler.org). Cytoskeletal fractions of protein were separated from unbound cytoplasmic fractions by rinsing with an ice cold high sucrose buffer for 1 min prior to fixation for immunoflourecence or MPER lysis buffer (Thermo) for western blotting. Significance is noted for p<0.05.
Results and Discussion: The integrins in randomly migrating human mesenchymal stem cells showed greater amounts of integrins on PMMA substrates than glass with nanofibers being slightly less than smooth substrates. Integrin beta3 significantly followed this trend. After washing out proteins not bound to the cytoskeleton in randomly migrating mouse embryonic fibroblasts, a greater proportion of available integrins were utilized on fibers verses the flat substrate control. Immunostaining shows that the integrins and focal adhesions (vinculin) localize along the fibers (Fig 1).
Attachment to nanofibers drives significant nuclear area reduction (p<0.05). The mean number of focal adhesions and the distance of those adhesions to the nucleus were both reduced by culture on fibers compared to glass (p<0.05), suggesting that more focal adhesions formed in the interior of the cell footprint when cells were cultured on fibers (Fig 2). Comparing the smaller to the larger fibers, a decrease in diameter produced a decreased vinculin staining intensity. Combined this suggests that smaller fibers promote more numerous less mature adhesions.
Conclusion: Vinculin positive adhesions form on thinner fibrous substrates closer to the nucleus and are less mature compared to larger fibers and have increased integrin content compared to glass. In future experiments we hypothesize that migration speed will be increased on fibers compared to flat substrates, and will increase with a decrease in fiber diameter. Further study of the focal adhesion initiated signaling cascades will inform the design of scaffolds using fiber diameter to control cell differentiation.
References:
[1] Pakorn Kanchanawong, Gleb Shtengel, Ana M. Pasapera, Ericka B. Ramko, Michael W. Davidson, Harald F. Hess, and Clare M. Waterman. Nanoscale architecture of integrin-based cell adhesions. Nature. 2010 Nov 25; 468(7323): 580–584. doi: 10.1038/nature09621.
[2] Roman Lutz, Kristopher Pataky, Neha Gadhari, Mattia Marelli, Juergen Brugger, and Matthias Chiquet. Nano-stenciled RGD-gold patterns that inhibit focal contact maturation induce lamellipodia formation in fibroblasts. PLoS One. 2011;6(9):e25459. doi: 10.1371/journal.pone.0025459.